http://2008.igem.org/wiki/index.php?title=Special:Contributions/Norayucel&feed=atom&limit=50&target=Norayucel&year=&month=2008.igem.org - User contributions [en]2024-03-28T22:50:17ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Jamboree/Schedule/Practice_sessionsJamboree/Schedule/Practice sessions2008-10-11T21:37:00Z<p>Norayucel: /* Friday November 7 : Practice Talks sign-up sheet */</p>
<hr />
<div>== Friday November 7 : Practice Talks sign-up sheet ==<br />
<br />
<br />
Use this sign-up sheet to sign up for a slot on Friday night (November 7) to practice your talk. Note that there will NOT be any A/V (audio/visual) support on staff. All classrooms will be unlocked and you should use them and leave them as you found them. <br />
<br />
There are a limited number of time slots available so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room. This should, however, give you a chance to practice your talk in a new environment.<br />
<br />
Also, there will also be pre-registration available beginning at 6pm. Conference services will be on-site to pass out team registration boxes (see the [[Jamboree/Compete#Team_boxes | Jamboree compete]] page). <br />
<br />
<br />
(Pizza and refreshments will be available on a first-come first-serve basis)<br />
<br />
<br />
<html><br />
<link rel="stylesheet" href="http://parts.mit.edu/igem07/index.php?title=User:Macowell/schedule.css&action=raw&ctype=text/css"><br />
<table class="calendar"><h2 class="date"><a name="Friday Practice">Friday, November 7</a></h2><br />
<thead><br />
<tr><br />
<th width="15%">Time</th><br />
<th>room 123</th><br />
<th>room 124</th><br />
<th>room 141</th><br />
<th>room 144</th><br />
<th>room 155</th><br />
<th>room G449</th><br />
<th>room D463</th><br />
<th>room 261*</th><br />
<th>room 262*</th><br />
<th>room 346*</th><br />
<th>room 397*</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="even"><br />
<th>6:00p - 6:30p</th><br />
<td>KULeuven</td><br />
<td>B1</td><br />
<td>C1</td><br />
<td>D1</td><br />
<td>E1</td><br />
<td>F1</td><br />
<td>G1</td><br />
<td>H1</td><br />
<td>I1</td><br />
<td>J1</td><br />
<td>K1</td><br />
</tr><br />
<tr class="odd"><br />
<th>6:30p - 7:00p</th><br />
<td>Heidelberg</td><br />
<td>HKUSTers</td><br />
<td>C2</td><br />
<td>D2</td><br />
<td>E2</td><br />
<td>F2</td><br />
<td>G2</td><br />
<td>H2</td><br />
<td>I2</td><br />
<td>J2</td><br />
<td>K2</td><br />
</tr><br />
<tr class="even"><br />
<th>7:00p - 7:30p</th><br />
<td>Warsaw</td><br />
<td>UVA</td><br />
<td>UChicago</td><br />
<td>D3</td><br />
<td>NYMU-Taipei</td><br />
<td>F3</td><br />
<td>G3</td><br />
<td>H3</td><br />
<td>I3</td><br />
<td>J3</td><br />
<td>K3</td><br />
</tr><br />
<tr class="even"><br />
<th>7:30p - 8:00p</th><br />
<td>UCSF</td><br />
<td>Peking</td><br />
<td>C4</td><br />
<td>D4</td><br />
<td>E4</td><br />
<td>F4</td><br />
<td>G4</td><br />
<td>H4</td><br />
<td>I4</td><br />
<td>J4</td><br />
<td>K4</td><br />
</tr><br />
<tr class="odd"><br />
<th>8:00p - 8:30p</th><br />
<td>Caltech</td><br />
<td>Tsinghua</td><br />
<td>C5</td><br />
<td>D5</td><br />
<td>E5</td><br />
<td>F5</td><br />
<td>G5</td><br />
<td>H5</td><br />
<td>I5</td><br />
<td>J5</td><br />
<td>K5</td><br />
</tr><br />
<tr class="even"><br />
<th>8:30p - 9:00p</th><br />
<td>A6</td><br />
<td>Alberta_NINT</td><br />
<td>ULeth</td><br />
<td>D6</td><br />
<td>E6</td><br />
<td>F6</td><br />
<td>G6</td><br />
<td>H6</td><br />
<td>I6</td><br />
<td>J6</td><br />
<td>K6</td><br />
</tr><br />
<tr class="odd"><br />
<th>9:00p - 9:30p</th><br />
<td>A7</td><br />
<td>Waterloo</td><br />
<td>Lethbridge_CCS</td><br />
<td>D7</td><br />
<td>E7</td><br />
<td>F7</td><br />
<td>G7</td><br />
<td>H7</td><br />
<td>I7</td><br />
<td>J7</td><br />
<td>K7</td><br />
</tr><br />
<tr class="even"><br />
<th>9:30p - 10:00p</th><br />
<td>A8</td><br />
<td>B8</td><br />
<td>C8</td><br />
<td>D8</td><br />
<td>E8</td><br />
<td>F8</td><br />
<td>G8</td><br />
<td>H8</td><br />
<td>I8</td><br />
<td>J8</td><br />
<td>K8</td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br />
<br />
Note that rooms marked with an asterisk (*) are smaller conference rooms throughout the Stata Center. Saturday sessions will not be held in these rooms but in order to accommodate all teams who would like to practice their presentations in the 4-hour period on Friday night, we must open these rooms for practice sessions.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:42:41Z<p>Norayucel: /* August 27, 2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
*Overnight culture didn’t grow, however plate has a few colonies<br />
*Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
*MINIPRPEP!<br />
<br />
*Stabs arrived from E0240 and OompX<br />
*Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene? Maybe think about that later!<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25, 2008==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
-Lane 1: 1kb ladder<br />
-Lane 2: Plasmid stock 1 <br />
-Lane 3: Plasmid stock 2<br />
-Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26, 2008==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5microL DNA/1microL dye.<br />
**Very strong bands<br />
<br />
*Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
<br />
-Enzyme EcoRI HindIII SolI BamHI<br />
-Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
-DNA (L) Buffer (B or H, microL) MilliQ water (microL) Enzyme (microL)<br />
-Single Digest 2.5 2 15 0.5<br />
-EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
-EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
*5 microL rxn mixture/1microL loading dye<br />
*15microL ladder<br />
-Lane 1 2 3 4 5 6 7 8<br />
-Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI<br />
<br />
==August 27, 2008==<br />
<br />
*Two bands for SolI, strange cuts in EcoRI/Sol.<br />
*Stocks 1 and 2 are the same.<br />
*Redo. Try old SolI stock and a new aliquot. <br />
*Send to sequencing with M13 primers<br />
<br />
- DNA (microL) Buffer (H, microL) MilliQ water (microL) Enzyme (microL)<br />
-Single Digest 1.5 2 16 0.5<br />
-EcoRI/SolI 8 2 (H) 9 0.5+0.5<br />
<br />
<br />
-Lane 1 2 3 4 5 6 7<br />
-Contents 1kb ladder NO enzyme SolI (old) SolI (new) EcoRI/SolI (old) EcoRI/SolI (new) pBluescript (check)<br />
<br />
*do Restriction digest with GFP and OmpX?<br />
<br />
==August 28, 2008==<br />
PC-Bio presentation!<br />
*442micrograms/microliter concentration (Stock 2 of pUC8 mystery plasmid)<br />
*No explanation as to why SolI is cutting twice. Submit to sequencing facility using M13Rev primer<br />
<br />
==August 29, 2008==<br />
Made BL21 competent cells with Rob (for pLysS). ~60 50microliters vials.<br />
<br />
==August 30, 2008==<br />
*50microliters cells/1 microliter pGreen<br />
*Add 100microliters SOC after electroporating, shake at 37C for one hour<br />
*Plate 80microliters on plates<br />
<br />
*Test transformation efficiency<br />
*On LB+Amp plates<br />
**BL21+10pg/microliter pGreen: 20microliters<br />
**BL21+100pg/microliter pGreen: 20 microliters<br />
**BL21 --: 20 microliters<br />
*On LB plates<br />
**BL21 --: 20 microliters<br />
<br />
==August 31, 2008==<br />
*Number of colonies<br />
**100pg: 100 colonies,<br />
***Efficiency is 1.3x10^7 transformants/microgram of DNA<br />
**10pg had only ~3 colonies (?)<br />
**No plasmid: Grew up on LB, didn’t on amp (so that checks out)<br />
<br />
==Sept 2, 2008==<br />
*Checked extra pUC8. Prep was fine.<br />
*Sequencing results show plasmid is pUC8 CVX. Whoo!<br />
<br />
==Sept 3, 2008==<br />
iGEM inventory (sent 8/28)<br />
*I20260 K –Measure ment kit test of J23101. (promoter +GFP, standard promoter)<br />
*I20269 K—J23150 (weak) promoter+GFP<br />
*I20270 K--<br />
*P1010-pSB3K3<br />
<br />
*J23101 A<br />
*P1010—pSB1A2<br />
<br />
==Sept 5, ,2008==<br />
*GROWING CAULOBACTER!<br />
<br />
*5ml O/N culture of pUC8 CVX transformed bacteria from damon’s plate’s (july 23). <br />
#: 100X dilution, with loop<br />
#:100X dilution, with pipette tip<br />
#:10X dilution, with loop<br />
#:10X dilution, with pipette tip.<br />
<br />
Tried own transformation with sequenced S2, pUC8 CVX DNA. <br />
*3microliters of DNA in 50microliters competent cells.<br />
*Time constant was 4.84, 4.90 for + and – plasmid respectively<br />
*Added 100microliters PYE following transformation, shook at 30C for 2.5 hours<br />
-Plate 1: PYE/Chlr. Caulobacter+pUC8, 100microliters<br />
-Plate 2: PYE/Chlr. Caulobacter+pUC8, 10microliters<br />
-Plate 3: PYE/Chlr. NO plasmid<br />
-Plate 4: PYE NO plasmid<br />
*Come back tomorrow morning ~8 to pick up culture/plates.<br />
<br />
*Rob is making media necessary for growth. Yay Rob!</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:41:38Z<p>Norayucel: /* August 26, 2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
*Overnight culture didn’t grow, however plate has a few colonies<br />
*Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
*MINIPRPEP!<br />
<br />
*Stabs arrived from E0240 and OompX<br />
*Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene? Maybe think about that later!<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25, 2008==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
-Lane 1: 1kb ladder<br />
-Lane 2: Plasmid stock 1 <br />
-Lane 3: Plasmid stock 2<br />
-Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26, 2008==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5microL DNA/1microL dye.<br />
**Very strong bands<br />
<br />
*Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
<br />
-Enzyme EcoRI HindIII SolI BamHI<br />
-Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
-DNA (L) Buffer (B or H, microL) MilliQ water (microL) Enzyme (microL)<br />
-Single Digest 2.5 2 15 0.5<br />
-EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
-EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
*5 microL rxn mixture/1microL loading dye<br />
*15microL ladder<br />
-Lane 1 2 3 4 5 6 7 8<br />
-Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI<br />
<br />
==August 27, 2008==<br />
<br />
*Two bands for SolI, strange cuts in EcoRI/Sol.<br />
*Stocks 1 and 2 are the same.<br />
*Redo. Try old SolI stock and a new aliquot. <br />
*Send to sequencing with M13 primers<br />
<br />
-DNA (microL) Buffer (H, microL) MilliQ water (microL) Enzyme (microL)<br />
-Single Digest 1.5 2 16 0.5<br />
-EcoRI/SolI 8 2 (H) 9 0.5+0.5<br />
<br />
<br />
-Lane 1 2 3 4 5 6 7<br />
-Contents 1kb ladder NO enzyme SolI (old) SolI (new) EcoRI/SolI (old) EcoRI/SolI (new) pBluescript (check)<br />
<br />
<br />
*do Restriction digest with GFP and OmpX?<br />
<br />
==August 28, 2008==<br />
PC-Bio presentation!<br />
*442micrograms/microliter concentration (Stock 2 of pUC8 mystery plasmid)<br />
*No explanation as to why SolI is cutting twice. Submit to sequencing facility using M13Rev primer<br />
<br />
==August 29, 2008==<br />
Made BL21 competent cells with Rob (for pLysS). ~60 50microliters vials.<br />
<br />
==August 30, 2008==<br />
*50microliters cells/1 microliter pGreen<br />
*Add 100microliters SOC after electroporating, shake at 37C for one hour<br />
*Plate 80microliters on plates<br />
<br />
*Test transformation efficiency<br />
*On LB+Amp plates<br />
**BL21+10pg/microliter pGreen: 20microliters<br />
**BL21+100pg/microliter pGreen: 20 microliters<br />
**BL21 --: 20 microliters<br />
*On LB plates<br />
**BL21 --: 20 microliters<br />
<br />
==August 31, 2008==<br />
*Number of colonies<br />
**100pg: 100 colonies,<br />
***Efficiency is 1.3x10^7 transformants/microgram of DNA<br />
**10pg had only ~3 colonies (?)<br />
**No plasmid: Grew up on LB, didn’t on amp (so that checks out)<br />
<br />
==Sept 2, 2008==<br />
*Checked extra pUC8. Prep was fine.<br />
*Sequencing results show plasmid is pUC8 CVX. Whoo!<br />
<br />
==Sept 3, 2008==<br />
iGEM inventory (sent 8/28)<br />
*I20260 K –Measure ment kit test of J23101. (promoter +GFP, standard promoter)<br />
*I20269 K—J23150 (weak) promoter+GFP<br />
*I20270 K--<br />
*P1010-pSB3K3<br />
<br />
*J23101 A<br />
*P1010—pSB1A2<br />
<br />
==Sept 5, ,2008==<br />
*GROWING CAULOBACTER!<br />
<br />
*5ml O/N culture of pUC8 CVX transformed bacteria from damon’s plate’s (july 23). <br />
#: 100X dilution, with loop<br />
#:100X dilution, with pipette tip<br />
#:10X dilution, with loop<br />
#:10X dilution, with pipette tip.<br />
<br />
Tried own transformation with sequenced S2, pUC8 CVX DNA. <br />
*3microliters of DNA in 50microliters competent cells.<br />
*Time constant was 4.84, 4.90 for + and – plasmid respectively<br />
*Added 100microliters PYE following transformation, shook at 30C for 2.5 hours<br />
-Plate 1: PYE/Chlr. Caulobacter+pUC8, 100microliters<br />
-Plate 2: PYE/Chlr. Caulobacter+pUC8, 10microliters<br />
-Plate 3: PYE/Chlr. NO plasmid<br />
-Plate 4: PYE NO plasmid<br />
*Come back tomorrow morning ~8 to pick up culture/plates.<br />
<br />
*Rob is making media necessary for growth. Yay Rob!</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:32:11Z<p>Norayucel: /* August 26, 2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
*Overnight culture didn’t grow, however plate has a few colonies<br />
*Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
*MINIPRPEP!<br />
<br />
*Stabs arrived from E0240 and OompX<br />
*Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene? Maybe think about that later!<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25, 2008==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
-Lane 1: 1kb ladder<br />
-Lane 2: Plasmid stock 1 <br />
-Lane 3: Plasmid stock 2<br />
-Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26, 2008==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5microL DNA/1microL dye.<br />
**Very strong bands<br />
<br />
*Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
<br />
-Enzyme EcoRI HindIII SolI BamHI<br />
-Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
-DNA (L) Buffer (B or H, microL) MilliQ water (microL) Enzyme (microL)<br />
-Single Digest 2.5 2 15 0.5<br />
-EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
-EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
*5 microL rxn mixture/1microL loading dye<br />
*15microL ladder<br />
-Lane 1 2 3 4 5 6 7 8<br />
-Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:30:49Z<p>Norayucel: /* August 26, 2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
*Overnight culture didn’t grow, however plate has a few colonies<br />
*Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
*MINIPRPEP!<br />
<br />
*Stabs arrived from E0240 and OompX<br />
*Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene? Maybe think about that later!<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25, 2008==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
-Lane 1: 1kb ladder<br />
-Lane 2: Plasmid stock 1 <br />
-Lane 3: Plasmid stock 2<br />
-Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26, 2008==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5microL DNA/1microL dye.<br />
**Very strong bands<br />
<br />
*Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
<br />
-Enzyme EcoRI HindIII SolI BamHI<br />
-Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
-DNA (L) Buffer (B or H, microL) MilliQ water (microL) Enzyme (microL)<br />
-Single Digest 2.5 2 15 0.5<br />
-EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
-EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
*5 microL rxn mixture/1microL loading dye<br />
*15microL ladder<br />
-Lane 1 2 3 4 5 6 7 8<br />
-Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:29:46Z<p>Norayucel: /* August 26 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
*Overnight culture didn’t grow, however plate has a few colonies<br />
*Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
*MINIPRPEP!<br />
<br />
*Stabs arrived from E0240 and OompX<br />
*Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene? Maybe think about that later!<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25, 2008==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
-Lane 1: 1kb ladder<br />
-Lane 2: Plasmid stock 1 <br />
-Lane 3: Plasmid stock 2<br />
-Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26, 2008==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5microL DNA/1microL dye.<br />
**Very strong bands<br />
<br />
*Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
<br />
-Enzyme EcoRI HindIII SolI BamHI<br />
-Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
-DNA (L) Buffer (B or H, microL) MilliQ water (microL) Enzyme (microL)<br />
-Single Digest 2.5 2 15 0.5<br />
-EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
-EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
*5 microL rxn mixture/1microL loading dye<br />
*15microL ladder<br />
-Lane 1 2 3 4 5 6 7 8<br />
-Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:28:11Z<p>Norayucel: /* August 25 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
*Overnight culture didn’t grow, however plate has a few colonies<br />
*Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
*MINIPRPEP!<br />
<br />
*Stabs arrived from E0240 and OompX<br />
*Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene? Maybe think about that later!<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25, 2008==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
-Lane 1: 1kb ladder<br />
-Lane 2: Plasmid stock 1 <br />
-Lane 3: Plasmid stock 2<br />
-Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
*Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
<br />
*Enzyme EcoRI HindIII SolI BamHI<br />
*Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
**DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
*Single Digest 2.5 2 15 0.5<br />
*EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
*EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
*5 L rxn mixture/1L loading dye<br />
*15L ladder<br />
*Lane 1 2 3 4 5 6 7 8<br />
*Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:27:16Z<p>Norayucel: /* August 26 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
*Overnight culture didn’t grow, however plate has a few colonies<br />
*Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
*MINIPRPEP!<br />
<br />
*Stabs arrived from E0240 and OompX<br />
*Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene? Maybe think about that later!<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
Lane 1: 1kb ladder<br />
Lane 2: Plasmid stock 1 <br />
Lane 3: Plasmid stock 2<br />
Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
*Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
<br />
*Enzyme EcoRI HindIII SolI BamHI<br />
*Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
**DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
*Single Digest 2.5 2 15 0.5<br />
*EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
*EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
*5 L rxn mixture/1L loading dye<br />
*15L ladder<br />
*Lane 1 2 3 4 5 6 7 8<br />
*Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:25:58Z<p>Norayucel: /* August 26 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
*Overnight culture didn’t grow, however plate has a few colonies<br />
*Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
*MINIPRPEP!<br />
<br />
*Stabs arrived from E0240 and OompX<br />
*Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene? Maybe think about that later!<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
Lane 1: 1kb ladder<br />
Lane 2: Plasmid stock 1 <br />
Lane 3: Plasmid stock 2<br />
Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
*Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
<br />
<br />
*Enzyme EcoRI HindIII SolI BamHI<br />
*Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
Single Digest 2.5 2 15 0.5<br />
EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
-5 L rxn mixture/1L loading dye<br />
-15L ladder<br />
Lane 1 2 3 4 5 6 7 8<br />
Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:24:33Z<p>Norayucel: /* August 22, 2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
*Overnight culture didn’t grow, however plate has a few colonies<br />
*Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
*MINIPRPEP!<br />
<br />
*Stabs arrived from E0240 and OompX<br />
*Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene? Maybe think about that later!<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
Lane 1: 1kb ladder<br />
Lane 2: Plasmid stock 1 <br />
Lane 3: Plasmid stock 2<br />
Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
Enzyme EcoRI HindIII SolI BamHI<br />
Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
Single Digest 2.5 2 15 0.5<br />
EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
-5 L rxn mixture/1L loading dye<br />
-15L ladder<br />
Lane 1 2 3 4 5 6 7 8<br />
Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:23:52Z<p>Norayucel: /* August 20,2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
*DH5alpha at competency of ~5x10^7 (15 colonies)<br />
*Still no iGEM plasmids. What gives?<br />
*Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
**iGEM at 80 picograms/microgram, control is at 72<br />
*Add one microliter of solution of 10microliters to 50 microliters DNA<br />
*Electroporate<br />
*All time constants between 4.6 and 4.9.<br />
<br />
==August 21, 2008==<br />
*XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
*iGEM still doesn’t transform. What gives?! <br />
*Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.<br />
*Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
*streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
==August 22, 2008==<br />
-Overnight culture didn’t grow, however plate has a few colonies<br />
-Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
-MINIPRPEP!<br />
<br />
-Stabs arrived from E0240 and OompX<br />
-Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
Lane 1: 1kb ladder<br />
Lane 2: Plasmid stock 1 <br />
Lane 3: Plasmid stock 2<br />
Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
Enzyme EcoRI HindIII SolI BamHI<br />
Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
Single Digest 2.5 2 15 0.5<br />
EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
-5 L rxn mixture/1L loading dye<br />
-15L ladder<br />
Lane 1 2 3 4 5 6 7 8<br />
Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:22:54Z<p>Norayucel: /* August 11, 2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
*Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
*Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)<br />
<br />
<br />
August 13/14 at PCBio retreat. Whoo!<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
-DH5alpha at competency of ~5x10^7 (15 colonies)<br />
-Still no iGEM plasmids. What gives?<br />
-Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
-iGEM at 80 picograms/microgram, control is at 72<br />
-Add one microliter of solution of 10microliters to 50 microliters DNA<br />
-Electroporate<br />
-All time constants between 4.6 and 4.9.<br />
<br />
August 21<br />
-XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
-iGEM still doesn’t transform. What gives?! <br />
-Check caulobacter: it’s caulobacter!!! <br />
-Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
-streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
<br />
==August 22, 2008==<br />
-Overnight culture didn’t grow, however plate has a few colonies<br />
-Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
-MINIPRPEP!<br />
<br />
-Stabs arrived from E0240 and OompX<br />
-Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
Lane 1: 1kb ladder<br />
Lane 2: Plasmid stock 1 <br />
Lane 3: Plasmid stock 2<br />
Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
Enzyme EcoRI HindIII SolI BamHI<br />
Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
Single Digest 2.5 2 15 0.5<br />
EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
-5 L rxn mixture/1L loading dye<br />
-15L ladder<br />
Lane 1 2 3 4 5 6 7 8<br />
Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:22:06Z<p>Norayucel: /* August 8, 2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
*TOP10+E2040/Amp: Nothing<br />
*TOP10+CFP/Amp:Nothing<br />
*TOP10/LB: Lots of cells (so cells are viable)<br />
*TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
-Made new amp plates with 250mL agar LB +250microliters of<br />
100mg/mL ampicillin (fresh)<br />
<br />
Transformations<br />
<br />
August 13/14 at retreat<br />
__________<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
-DH5alpha at competency of ~5x10^7 (15 colonies)<br />
-Still no iGEM plasmids. What gives?<br />
-Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
-iGEM at 80 picograms/microgram, control is at 72<br />
-Add one microliter of solution of 10microliters to 50 microliters DNA<br />
-Electroporate<br />
-All time constants between 4.6 and 4.9.<br />
<br />
August 21<br />
-XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
-iGEM still doesn’t transform. What gives?! <br />
-Check caulobacter: it’s caulobacter!!! <br />
-Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
-streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
<br />
==August 22, 2008==<br />
-Overnight culture didn’t grow, however plate has a few colonies<br />
-Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
-MINIPRPEP!<br />
<br />
-Stabs arrived from E0240 and OompX<br />
-Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
Lane 1: 1kb ladder<br />
Lane 2: Plasmid stock 1 <br />
Lane 3: Plasmid stock 2<br />
Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
Enzyme EcoRI HindIII SolI BamHI<br />
Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
Single Digest 2.5 2 15 0.5<br />
EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
-5 L rxn mixture/1L loading dye<br />
-15L ladder<br />
Lane 1 2 3 4 5 6 7 8<br />
Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:21:10Z<p>Norayucel: /* August 7,2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
*TOP10+p1010/LB: Lots of cells<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): 12 very small green colonies<br />
*TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
*TOP10+E2040/Amp<br />
*TOP10+CFP/Amp<br />
*TOP10/LB:<br />
*TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
TOP10+E2040/Amp: Nothing<br />
TOP10+CFP/Amp:Nothing<br />
TOP10/LB:: Lots of cells (so cells are viable)<br />
TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
-Made new amp plates with 250mL agar LB +250microliters of<br />
100mg/mL ampicillin (fresh)<br />
<br />
Transformations<br />
<br />
August 13/14 at retreat<br />
__________<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
-DH5alpha at competency of ~5x10^7 (15 colonies)<br />
-Still no iGEM plasmids. What gives?<br />
-Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
-iGEM at 80 picograms/microgram, control is at 72<br />
-Add one microliter of solution of 10microliters to 50 microliters DNA<br />
-Electroporate<br />
-All time constants between 4.6 and 4.9.<br />
<br />
August 21<br />
-XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
-iGEM still doesn’t transform. What gives?! <br />
-Check caulobacter: it’s caulobacter!!! <br />
-Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
-streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
<br />
==August 22, 2008==<br />
-Overnight culture didn’t grow, however plate has a few colonies<br />
-Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
-MINIPRPEP!<br />
<br />
-Stabs arrived from E0240 and OompX<br />
-Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
Lane 1: 1kb ladder<br />
Lane 2: Plasmid stock 1 <br />
Lane 3: Plasmid stock 2<br />
Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
Enzyme EcoRI HindIII SolI BamHI<br />
Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
Single Digest 2.5 2 15 0.5<br />
EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
-5 L rxn mixture/1L loading dye<br />
-15L ladder<br />
Lane 1 2 3 4 5 6 7 8<br />
Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:20:43Z<p>Norayucel: /* August 6, 2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
*+pGreen 10microliters: haze, with a few blips that are not green<br />
*+pGreen 50 microliters: haze, more blips, but still not green<br />
*+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
*-plasmid: Nothing. <br />
*+pGreen 50 microliters: nothing<br />
<br />
LB<br />
*-plasmid: nothing<br />
*+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
*TOP10+p1010/LB: smear<br />
*TOP10+E02040/Amp: nothing<br />
*TOP10+pGreen (125microliters): ~20 colonies<br />
*TOP10+TE buffer: smear<br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
TOP10+p1010/LB: Lots of cells<br />
TOP10+E02040/Amp: nothing<br />
TOP10+pGreen (125microliters): 12 very small green colonies<br />
TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
TOP10+E2040/Amp<br />
TOP10+CFP/Amp<br />
TOP10/LB:<br />
TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
TOP10+E2040/Amp: Nothing<br />
TOP10+CFP/Amp:Nothing<br />
TOP10/LB:: Lots of cells (so cells are viable)<br />
TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
-Made new amp plates with 250mL agar LB +250microliters of<br />
100mg/mL ampicillin (fresh)<br />
<br />
Transformations<br />
<br />
August 13/14 at retreat<br />
__________<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
-DH5alpha at competency of ~5x10^7 (15 colonies)<br />
-Still no iGEM plasmids. What gives?<br />
-Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
-iGEM at 80 picograms/microgram, control is at 72<br />
-Add one microliter of solution of 10microliters to 50 microliters DNA<br />
-Electroporate<br />
-All time constants between 4.6 and 4.9.<br />
<br />
August 21<br />
-XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
-iGEM still doesn’t transform. What gives?! <br />
-Check caulobacter: it’s caulobacter!!! <br />
-Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
-streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
<br />
==August 22, 2008==<br />
-Overnight culture didn’t grow, however plate has a few colonies<br />
-Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
-MINIPRPEP!<br />
<br />
-Stabs arrived from E0240 and OompX<br />
-Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
Lane 1: 1kb ladder<br />
Lane 2: Plasmid stock 1 <br />
Lane 3: Plasmid stock 2<br />
Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
Enzyme EcoRI HindIII SolI BamHI<br />
Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
Single Digest 2.5 2 15 0.5<br />
EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
-5 L rxn mixture/1L loading dye<br />
-15L ladder<br />
Lane 1 2 3 4 5 6 7 8<br />
Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/NorayucelTeam:University of Chicago/Notebook/Norayucel2008-09-28T16:18:54Z<p>Norayucel: /* July 29, 2008 */</p>
<hr />
<div>==July 18, 2008==<br />
<br />
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution<br><br />
2. 1microliter of plasmid to 50 microliters competent cells<br><br />
3. Will grow up 50microliters of TOP10 untransformed as a control<br><br />
4. Put on ice at 2:03<br><br />
5. Took off ice at 2:34<br><br />
6. Incubated for 1minute at 42C<br><br />
7. Added 250microliters SOC media (prepared at 1:30)<br><br />
8. Start incubated at 37C at 2:36<br><br />
9. Take out at 3:40pm<br><br />
10. Streaked 20microliters onto Amp. Plates<br><br />
*+pGreen<br />
*NO plasmid<br />
11. Dan will come in Saturday morning to pick up the plates and count colonies.<br><br />
<br />
==July 21, 2008==<br />
<br />
#No colonies grew on 10pg/microliter.<br />
#Try transformation again before trying to grow up new TOP10<br />
#*DNA could be bad<br />
#*Wrong antibiotic (double checked: ampicillin is correct)<br />
#*Something went wrong with transformation--perhaps too long at 42C?<br />
#Bad cells (nooooo....)<br><br />
'''Retry'''<br />
*10pg/microliter<br />
*100pg/microliter<br />
*1ng/microliter<br />
*no plasmid on LB (test if cells are completely dead)<br />
'''Also try Dr. Schonbaum's stocks to compare'''<br />
*10pg/microliter<br />
*1ng/microliter<br><br />
transformed at 4pm and put in 37C incubator. Check tomorrow morning.<br />
<br />
==July 22, 2008==<br />
*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006.<br />
#Will track OD during day until Damon comes in<br />
#*9:45 OD:0.006<br />
#*11:52: 0.017<br />
#*1:50pm: 0.033<br />
#Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.<br />
<br />
'''TOP 10'''<br />
#10pg: 3 colonies.<br />
#100pg: 25 colonies<br />
#1ng: 200 colonies<br />
*Efficiency of cells is ~3x10^6. Need to redo.<br />
#NO plasmid on LB: big streaky mess. Cell mos' def' alive.<br />
#STOCKS, 10pg: 22<br />
#Stocks, 1ng: 300-400<br />
#*Weird. Should be 100X more than 10pg. Perhaps bad dilution.<br />
#*Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....<br />
<br />
==July 23, 2008==<br />
*Damon needs the shakers at 30C, so will have to put off redo of TOP10.<br />
'''Check OD'''<br />
#10:00am: OD is 1.43.<br />
#10:45: OD is .14.<br />
#*Dilute to .14<br />
#*~700mL is 2L flask<br />
#1:45pm: OD is .39<br />
<br />
==July 24, 2008==<br />
#Set 3mL starter culture at ~6pm.<br />
<br />
==July 25, 2008==<br />
#Remade TOP10 competent cells--will try to make more competent<br />
#Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture<br />
#*Two 2L flasks with 250mL each<br />
#At 5:30 OD was .28 and .29 for each flask respectively<br />
#Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)<br />
#Put into -80C at 7:30pm.<br />
<br />
==July 28, 2008==<br />
'''Transformation with pGreen''' <br />
#10pg/microliter on LB Amp<br />
#1 ng/microliter on LB Amp<br />
#NO plasmid on LB Amp<br />
#NO plasmid on plain LB<br />
#Put into 37C incubator ~1:30pm<br><br><br />
'''Plates'''<br />
#Running out of plates<br />
# 500mL of LB and LB Amp agar<br />
#*100mg/mL stock of Ampicillan<br />
#* After cooling for ~45 minutes at room temp, added .5mL<br />
#Poured around 4:30 pm. Turned over at 5:30<br><br />
<br />
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning<br />
<br />
==July 29, 2008==<br />
#Checked plates<br />
# 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(<br />
#1ng/microliter plate was a big streaky mess (too many to count)<br />
#NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??<br />
#NO plasmid +LB big streaky mess (as expected)<br />
<br />
==August 5, 2008==<br />
Transformations with DH5alpha<br />
<br />
Old LB/Amp plates<br />
-plasmid: 200microliters<br />
+pGreen: 10microliters<br />
+pGreen: 50 microliters<br />
+pGreen: 250 microliters<br />
<br />
New LB/Amp plates<br />
-plasmid: 200 microliters<br />
+pGreen: 50 microliters<br />
<br />
LB<br />
-plasmid<br />
+pGreen<br />
<br />
==August 6, 2008==<br />
<br />
RESULTS<br />
<br />
DH5-ALPHA (plated on 8.5.08)<br />
Old LB/Amp plates<br />
-strange film on all the old LB/Amp plates. Contamination?<br />
-plasmid: Only haze<br />
+pGreen 10microliters: haze, with a few blips that are not green<br />
+pGreen 50 microliters: haze, more blips, but still not green<br />
+pGreen 250microliters: haze+non-green blips<br />
<br />
New LB/Amp plates<br />
-plasmid: Nothing. <br />
+pGreen 50 microliters: nothing<br />
<br />
LB<br />
-plasmid: nothing<br />
+pGreen 50 microliters: lots of cells<br />
<br />
Transformations iGEM:<br />
TOP10+p1010/LB<br />
TOP10+E02040/Amp:<br />
TOP10+pGreen (125microliters):<br />
TOP10+TE buffer: <br />
<br />
==August 7,2008==<br />
<br />
RESULTS from iGEM (8.6.08)<br />
TOP10+p1010/LB: Lots of cells<br />
TOP10+E02040/Amp: nothing<br />
TOP10+pGreen (125microliters): 12 very small green colonies<br />
TOP10+TE buffer:Lots of cells<br />
<br />
..Results don't look so hot. Lets try the transformation again. <br />
<br />
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp<br />
plates are bad?<br />
<br />
Plates<br />
TOP10+E2040/Amp<br />
TOP10+CFP/Amp<br />
TOP10/LB:<br />
TOP10+pGreen<br />
<br />
==August 8, 2008==<br />
RESULTS from 8.7.08 transformation<br />
TOP10+E2040/Amp: Nothing<br />
TOP10+CFP/Amp:Nothing<br />
TOP10/LB:: Lots of cells (so cells are viable)<br />
TOP10+pGreen: 40 colonies<br />
<br />
==August 11, 2008==<br />
Retry pGreen/DH5alpha with Schonbaum's Amp plates<br />
-Made new amp plates with 250mL agar LB +250microliters of<br />
100mg/mL ampicillin (fresh)<br />
<br />
Transformations<br />
<br />
August 13/14 at retreat<br />
__________<br />
<br />
==August 18==<br />
*Grew up 250mL of DH5-alpha in LB<br />
*Prepped using Thomas’s protocol<br />
*Grew for three hours, stopped at OD of .301<br />
*Total volume ~800microliters<br />
<br />
==August 19, 2008==<br />
Grew up 250mL of XL21 in SOB<br />
*had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew<br />
*Start shaking at 9:30, took out at 1:20. (OD of .35)<br />
*Electroporation of GFP generator with DH5alpha<br />
**pGreen (1microliter of 10pg/microliter)<br />
**E0240<br />
**Using stock cells+E0240<br />
*Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82<br />
*With E0240 time constant was 8.22 (?)<br />
*Stock cells popped—junked it.<br />
*Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)<br />
<br />
==August 20,2008==<br />
-DH5alpha at competency of ~5x10^7 (15 colonies)<br />
-Still no iGEM plasmids. What gives?<br />
-Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid<br />
-iGEM at 80 picograms/microgram, control is at 72<br />
-Add one microliter of solution of 10microliters to 50 microliters DNA<br />
-Electroporate<br />
-All time constants between 4.6 and 4.9.<br />
<br />
August 21<br />
-XL1 BLUE ARE COMPENTENT! ~320 colonies<br />
-iGEM still doesn’t transform. What gives?! <br />
-Check caulobacter: it’s caulobacter!!! <br />
-Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)<br />
-streaked one colony of mystery plasmid on chloramphenicol plate<br />
<br />
<br />
==August 22, 2008==<br />
-Overnight culture didn’t grow, however plate has a few colonies<br />
-Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.<br />
-MINIPRPEP!<br />
<br />
-Stabs arrived from E0240 and OompX<br />
-Streak<br />
cacc tagaatatca<br />
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc<br />
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt<br />
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt<br />
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag<br />
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac<br />
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa<br />
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat<br />
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc<br />
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg<br />
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt<br />
661 gctcaggcgg ccagaatatt aggaagggct aaataatta<br />
<br />
Order:<br />
LL-H Lysin/thymol<br />
Princeton apoptosis gene<br />
<br />
==August 23, 2008==<br />
-Both stocks of pUC8 have cells. Whoo!<br />
-How to deal with bacterial stab?<br />
<br />
==August 24, 2008==<br />
-Put stabs in 37C incubator.<br />
<br />
==August 25==<br />
*Miniprep DNA, digest with BamHI and EcoRI<br />
*Run gel<br />
Lane 1: 1kb ladder<br />
Lane 2: Plasmid stock 1 <br />
Lane 3: Plasmid stock 2<br />
Lane 4: DWP1 (miniprepped plasmid)<br />
*band. Plasmid definitely present.<br />
*3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.<br />
*Create biobricks for mefp-5 caulobacter and E. coli<br />
<br />
==August 26==<br />
*both iGEM stocks grew up<br />
*GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)<br />
**Ran on 1.2% agarose gel. 5L DNA/1L dye.<br />
**Very strong bands<br />
<br />
Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.<br />
*Use EcoRI, HindIII, SolI, BamHI<br />
<br />
Buffers<br />
Enzyme EcoRI HindIII SolI BamHI<br />
Buffer (10x) H B H B<br />
<br />
Materials/amounts<br />
DNA (L) Buffer (B or H, L) MilliQ water (L) Enzyme (L)<br />
Single Digest 2.5 2 15 0.5<br />
EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5<br />
EcoRI/SolI 10 2 (H) 7 0.5+0.5<br />
<br />
<br />
Agarose Gels (for each stock, 1 or 2)<br />
-5 L rxn mixture/1L loading dye<br />
-15L ladder<br />
Lane 1 2 3 4 5 6 7 8<br />
Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/TransformationsTeam:University of Chicago/Notebook/Transformations2008-08-08T15:15:48Z<p>Norayucel: /* iGEM transformatin (using paper spots) */</p>
<hr />
<div>==Chemocompetent cells==<br />
<br />
===General transformation===<br />
#Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)<br />
#* This is at 10 pg/microliter or 10-5 _g/_l<br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours<br />
# Heat shock 60 sec at 42C<br />
# Add 250 _l SOC<br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated<br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.<br />
#* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies<br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads<br />
* Good cells should yield around 100 - 400 colonies<br />
* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA<br />
* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA<br />
<br />
===iGEM transformation (using paper spots)===<br />
'''Materials needed'''<br />
*Spots soaked in 5microliters warm TE for 20 minutes<br />
*2.0 conical bottom tubes<br />
*Ice<br />
*Competent cells<br />
*42C water bath/37C incubator<br />
*SOC--freshly made!<br />
*Petri plates with appropriate antibiotic<br />
*Transformaiton control DNA, like pGreen<br />
'''Procedure'''<br />
#Soak the spots in 5microliters of the warmed TE for 20 minutes. This allows the maximum concentration of DNA in solution. Start thawing the competent cells on wet crushed ice<br />
#Chill lageled 2mL conical bottom tubes on wet ice. Add 2 microliters of DNA in TE into 50microliters thawed TOP10 cells in 2mL tubes. 2mL tubes allow better liquid movement durin incubation. <br />
#*Make sure to do a control transformation! iGEM uses 1microliter of pUC19 plasmid DNA from invitrogen; we'll use pGreen from Dr. Schonbaum<br />
#Hold the DNA and competent cells on ice for 30 mintues. <br />
#Heat shock cells by immersion in a pre-heated water bath at 42C for 60 seconds. A water bath is important to improve heat transfer to the cells.<br />
# Incubate cell son ice for 2 minutes<br />
#Add 200microliters of SOC broth. This should have NO antibiotics.<br />
#Incubate cells at 37C for 2 hours while tubes are rotating/shaking. 2 hours growth helps in transfomation efficiency, especially for plasmids with resistance other than ampicillin.<br />
# LAbel an LB agar plate w/appropriate antibiotic(s) with the part number, plasmid, and resistance. Plate 250 microliters of the cell culture on the plate.<br />
#Incubate plate at 37C ofr 12-14 hours, making sure agar side is up. If incubated for too long, antibiotics, '''especially ampicillin''' to break down and untransformed cells will grow.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/TransformationsTeam:University of Chicago/Notebook/Transformations2008-08-08T15:15:31Z<p>Norayucel: /* iGEM transformatin (using paper spots) */</p>
<hr />
<div>==Chemocompetent cells==<br />
<br />
===General transformation===<br />
#Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)<br />
#* This is at 10 pg/microliter or 10-5 _g/_l<br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours<br />
# Heat shock 60 sec at 42C<br />
# Add 250 _l SOC<br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated<br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.<br />
#* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies<br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads<br />
* Good cells should yield around 100 - 400 colonies<br />
* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA<br />
* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA<br />
<br />
===iGEM transformatin (using paper spots)===<br />
'''Materials needed'''<br />
*Spots soaked in 5microliters warm TE for 20 minutes<br />
*2.0 conical bottom tubes<br />
*Ice<br />
*Competent cells<br />
*42C water bath/37C incubator<br />
*SOC--freshly made!<br />
*Petri plates with appropriate antibiotic<br />
*Transformaiton control DNA, like pGreen<br />
'''Procedure'''<br />
#Soak the spots in 5microliters of the warmed TE for 20 minutes. This allows the maximum concentration of DNA in solution. Start thawing the competent cells on wet crushed ice<br />
#Chill lageled 2mL conical bottom tubes on wet ice. Add 2 microliters of DNA in TE into 50microliters thawed TOP10 cells in 2mL tubes. 2mL tubes allow better liquid movement durin incubation. <br />
#*Make sure to do a control transformation! iGEM uses 1microliter of pUC19 plasmid DNA from invitrogen; we'll use pGreen from Dr. Schonbaum<br />
#Hold the DNA and competent cells on ice for 30 mintues. <br />
#Heat shock cells by immersion in a pre-heated water bath at 42C for 60 seconds. A water bath is important to improve heat transfer to the cells.<br />
# Incubate cell son ice for 2 minutes<br />
#Add 200microliters of SOC broth. This should have NO antibiotics.<br />
#Incubate cells at 37C for 2 hours while tubes are rotating/shaking. 2 hours growth helps in transfomation efficiency, especially for plasmids with resistance other than ampicillin.<br />
# LAbel an LB agar plate w/appropriate antibiotic(s) with the part number, plasmid, and resistance. Plate 250 microliters of the cell culture on the plate.<br />
#Incubate plate at 37C ofr 12-14 hours, making sure agar side is up. If incubated for too long, antibiotics, '''especially ampicillin''' to break down and untransformed cells will grow.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/TransformationsTeam:University of Chicago/Notebook/Transformations2008-08-08T15:15:03Z<p>Norayucel: /* iGEM transformatin (using paper spots) */</p>
<hr />
<div>==Chemocompetent cells==<br />
<br />
===General transformation===<br />
#Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)<br />
#* This is at 10 pg/microliter or 10-5 _g/_l<br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours<br />
# Heat shock 60 sec at 42C<br />
# Add 250 _l SOC<br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated<br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.<br />
#* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies<br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads<br />
* Good cells should yield around 100 - 400 colonies<br />
* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA<br />
* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA<br />
<br />
===iGEM transformatin (using paper spots)===<br />
'''Materials needed'''<br />
*Spots soaked in 5microliters warm TE for 20 minutes<br />
*2.0 conical bottom tubes<br />
*Ice<br />
*Competent cells<br />
*42C water bath/37C incubator<br />
*SOC--freshly made!<br />
*Petri plates with appropriate antibiotic<br />
*Transformaiton control DNA, like pGreen<br />
#Soack the spots in 5microliters of the warmed TE for 20 minutes. This allows the maximum concentration of DNA in solution. Start thawing the competent cells on wet crushed ice<br />
#Chill lageled 2mL conical bottom tubes on wet ice. Add 2 microliters of DNA in TE into 50microliters thawed TOP10 cells in 2mL tubes. 2mL tubes allow better liquid movement durin incubation. <br />
#*Make sure to do a control transformation! iGEM uses 1microliter of pUC19 plasmid DNA from invitrogen; we'll use pGreen from Dr. Schonbaum<br />
#Hold the DNA and competent cells on ice for 30 mintues. <br />
#Heat shock cells by immersion in a pre-heated water bath at 42C for 60 seconds. A water bath is important to improve heat transfer to the cells.<br />
# Incubate cell son ice for 2 minutes<br />
#Add 200microliters of SOC broth. This should have NO antibiotics.<br />
#Incubate cells at 37C for 2 hours while tubes are rotating/shaking. 2 hours growth helps in transfomation efficiency, especially for plasmids with resistance other than ampicillin.<br />
# LAbel an LB agar plate w/appropriate antibiotic(s) with the part number, plasmid, and resistance. Plate 250 microliters of the cell culture on the plate.<br />
#Incubate plate at 37C ofr 12-14 hours, making sure agar side is up. If incubated for too long, antibiotics, '''especially ampicillin''' to break down and untransformed cells will grow.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/TransformationsTeam:University of Chicago/Notebook/Transformations2008-08-08T15:14:27Z<p>Norayucel: /* General transformation */</p>
<hr />
<div>==Chemocompetent cells==<br />
<br />
===General transformation===<br />
#Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)<br />
#* This is at 10 pg/microliter or 10-5 _g/_l<br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours<br />
# Heat shock 60 sec at 42C<br />
# Add 250 _l SOC<br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated<br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.<br />
#* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies<br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads<br />
* Good cells should yield around 100 - 400 colonies<br />
* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA<br />
* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA<br />
<br />
===iGEM transformatin (using paper spots)===<br />
'''Materials needed'''<br />
*Spots soaked in 5microliters warm TE for 20 minutes<br />
*2.0 conical bottom tubes<br />
*Ice<br />
*Competent cells<br />
*42C water bath/37C incubator<br />
*SOC--freshly made!<br />
*Petri plates with appropriate antibiotic<br />
*Transformaiton control DNA, like pGreen<br />
#Soack the spots in 5microliters of the warmed TE for 20 minutes. This allows the maximum concentration of DNA in solution. Start thawing the competent cells on wet crushed ice<br />
#Chill lageled 2mL conical bottom tubes on wet ice. Add 2 microliters of DNA in TE into 50microliters thawed TOP10 cells in 2mL tubes. 2mL tubes allow better liquid movement durin incubation. <br />
#*Make sure to do a control transformation! iGEM uses 1microliter of pUC19 plasmid DNA from invitrogen; we'll use pGreen from Dr. Schonbaum<br />
#Hold the DNA and competent cells on ice for 30 mintues. <br />
#Heat shock cells by immersion in a pre-heated water bath at 42C for 60 seconds. A water bath is important to improve heat transfer to the cells.<br />
# Incubate cell son ice for 2 minutes<br />
#Add 200microliters of SOC broth. This should have NO antibiotics.<br />
#Incubate cells at 37C for 2 hours while tubes are rotating/shaking. 2 hours growth helps in transfomation efficiency, especially for plasmids with resistance other than ampicillin.<br />
# LAbel an LB agar plate w/appropriate antibiotic(s) with the part number, plasmid, and resistance. Plate 250 microliters of the cell culture on the plate.<br />
#Incubate plate at 37C ofr 12-14 hours, making sure agar side is up. If incubated for too long, antibiotics, '''especially ampicillin''' staProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
to break down and untransformed cells will grow.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/TransformationsTeam:University of Chicago/Notebook/Transformations2008-08-08T15:05:30Z<p>Norayucel: /* =General transformation */</p>
<hr />
<div>==Chemocompetent cells==<br />
<br />
===General transformation===<br />
#Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)<br />
#* This is at 10 pg/microliter or 10-5 _g/_l<br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours<br />
# Heat shock 60 sec at 42C<br />
# Add 250 _l SOC<br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated<br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.<br />
#* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies<br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads<br />
* Good cells should yield around 100 - 400 colonies<br />
* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA<br />
* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/TransformationsTeam:University of Chicago/Notebook/Transformations2008-08-08T15:05:08Z<p>Norayucel: /* =General transformation */</p>
<hr />
<div>==Chemocompetent cells==<br />
<br />
===General transformation==<br />
#Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)<br />
#* This is at 10 pg/microliter or 10-5 _g/_l<br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours<br />
# Heat shock 60 sec at 42C<br />
# Add 250 _l SOC<br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated<br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.<br />
#* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies<br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads<br />
* Good cells should yield around 100 - 400 colonies<br />
* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA<br />
* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/TransformationsTeam:University of Chicago/Notebook/Transformations2008-08-08T15:04:21Z<p>Norayucel: /* =General transformation */</p>
<hr />
<div>==Chemocompetent cells==<br />
<br />
===General transformation==<br />
#Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)<br />
* This is at 10 pg/_l or 10-5 _g/_l<br />
* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours<br />
# Heat shock 60 sec at 42C<br />
# Add 250 _l SOC<br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated<br />
* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.<br />
* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies<br />
* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads<br />
* Good cells should yield around 100 - 400 colonies<br />
* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA<br />
* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/TransformationsTeam:University of Chicago/Notebook/Transformations2008-08-08T15:03:49Z<p>Norayucel: New page: ==Chemocompetent cells== ===General transformation== #Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) * This is at 10 pg/_l or 10-5 _g/_l * This can be made by ...</p>
<hr />
<div>==Chemocompetent cells==<br />
<br />
===General transformation==<br />
#Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)<br />
* This is at 10 pg/_l or 10-5 _g/_l<br />
* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours<br />
# Heat shock 60 sec at 42C<br />
# Add 250 _l SOC<br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated<br />
* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.<br />
* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol<br />
and tetracycline resistant, we find growing for 2 hours yields many more colonies<br />
* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads<br />
* Good cells should yield around 100 - 400 colonies<br />
* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA<br />
* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-08-08T15:00:59Z<p>Norayucel: /* Protocols */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
* [[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]<br />
* [[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]<br />
<br />
Remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Materials ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
*[[Team:University_of_Chicago/Notebook/Plasmids|Plasmids]]<br />
<br />
== Protocols ==<br />
*[[Team:University_of_Chicago/Notebook/TOP10 competent cells|TOP10 competent cells]]<br />
*[[Team:University_of_Chicago/Notebook/DH5-alpha competent cells|DH5-alpha competent cells]]<br />
*[[Team:University_of_Chicago/Notebook/Transformations|Transformations]]<br />
*[[Team:University_of_Chicago/Notebook/Glycerol stocks|Glycerol stocks]]<br />
*[[Team:University_of_Chicago/Notebook/DNA mini-prep|DNA mini-prep]]<br />
*[http://www.jobinyvon.com/Raman/Tutorial-Intro Raman Spectroscopy Tutorial]<br />
*[http://www.afmuniversity.org/ AFM Tutorial]</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/DH5-alpha_competent_cellsTeam:University of Chicago/Notebook/DH5-alpha competent cells2008-08-07T16:18:17Z<p>Norayucel: /* Procedure */</p>
<hr />
<div>==Materials==<br />
<br />
===TB Solution===<br />
*10mM PIPES<br />
*15mM CaCl2<br />
*250mM KCl<br />
*Dissolve in nanopure water and adjust pH 6.7 with KOH or HCL (solutes will dissolve as you do this) and then add<br />
55mM MnCl2. Adjust to final volume. Sterilize by filtration with 0.45um filter and store at 4°C<br />
<br />
===LB Media===<br />
(per 1L batch)<br />
*30 g LB mix<br />
*Dissolve in ddH2O, filling to 1L. <br />
*for 20mM MgSO4 FIRST add 16mL 1M MgSO4 solution THEN fill to 1L<br />
<br />
<br />
==Procedure==<br />
# Inoculate a 3ml overnight of E.coli in LB+20 mM MgSO4.<br />
# Next morning, inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml<br />
overnight culture. Grow at room temp (23°C) with good aeration (250rpm) to an <br />
A600 of 0.4-0.6.<br />
*Note: TEMPERATURE IS IMPORTANT! At 37C cells will grow up to proper OD in ~3 hours. A faster growing time, however, compromises efficiency, so choose temperature accordingly.<br />
#Place cells 10 min on ice. Transfer to a sterile bottle and spin 3K, 10', 4°C.<br />
# Resuspend pellet in 80 ml cold TB (swirl cells in bottle). Leave 10’/ice.<br />
# Spin cells 3K, 10', 4°C.<br />
#Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO. Leave 10'/ice.<br />
# Dispense into 220 ul and 525 ul aliquots (in cold sterile tubes) and <br />
freeze in dry ice/EtOH bath. Store -70°C. Typically, competency about 5X 106<br />
cfu/ug DNA. Note, improves after freezing. Cells good for a year and counting.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/DH5-alpha_competent_cellsTeam:University of Chicago/Notebook/DH5-alpha competent cells2008-08-07T16:17:51Z<p>Norayucel: New page: ==Materials== ===TB Solution=== *10mM PIPES *15mM CaCl2 *250mM KCl *Dissolve in nanopure water and adjust pH 6.7 with KOH or HCL (solutes will dissolve as you do this) and then add 55mM M...</p>
<hr />
<div>==Materials==<br />
<br />
===TB Solution===<br />
*10mM PIPES<br />
*15mM CaCl2<br />
*250mM KCl<br />
*Dissolve in nanopure water and adjust pH 6.7 with KOH or HCL (solutes will dissolve as you do this) and then add<br />
55mM MnCl2. Adjust to final volume. Sterilize by filtration with 0.45um filter and store at 4°C<br />
<br />
===LB Media===<br />
(per 1L batch)<br />
*30 g LB mix<br />
*Dissolve in ddH2O, filling to 1L. <br />
*for 20mM MgSO4 FIRST add 16mL 1M MgSO4 solution THEN fill to 1L<br />
<br />
<br />
==Procedure==<br />
1. Inoculate a 3ml overnight of E.coli in LB+20 mM MgSO4.<br />
2. Next morning, inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml<br />
overnight culture. Grow at room temp (23°C) with good aeration (250rpm) to an <br />
A600 of 0.4-0.6.<br />
*Note: TEMPERATURE IS IMPORTANT! At 37C cells will grow up to proper OD in ~3 hours. A faster growing time, however, compromises efficiency, so choose temperature accordingly.<br />
3. Place cells 10 min on ice. Transfer to a sterile bottle and spin 3K, 10', 4°C.<br />
4. Resuspend pellet in 80 ml cold TB (swirl cells in bottle). Leave 10’/ice.<br />
5. Spin cells 3K, 10', 4°C.<br />
6. Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO. Leave 10'/ice.<br />
7. Dispense into 220 ul and 525 ul aliquots (in cold sterile tubes) and <br />
freeze in dry ice/EtOH bath. Store -70°C. Typically, competency about 5X 106<br />
cfu/ug DNA. Note, improves after freezing. Cells good for a year and counting.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-08-07T15:53:34Z<p>Norayucel: /* Protocols */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
* [[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]<br />
* [[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]<br />
<br />
Remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Materials ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
*[[Team:University_of_Chicago/Notebook/Plasmids|Plasmids]]<br />
<br />
== Protocols ==<br />
*[[Team:University_of_Chicago/Notebook/TOP10 competent cells|TOP10 competent cells]]<br />
*[[Team:University_of_Chicago/Notebook/DH5-alpha competent cells|DH5-alpha competent cells]]<br />
*[[Team:University_of_Chicago/Notebook/Glycerol stocks|Glycerol stocks]]<br />
*[[Team:University_of_Chicago/Notebook/DNA mini-prep|DNA mini-prep]]</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/PlasmidsTeam:University of Chicago/Notebook/Plasmids2008-07-29T19:14:21Z<p>Norayucel: /* pBluescript SK */</p>
<hr />
<div>==pBluescript SK==<br />
[http://www.addgene.org/pgvec1?f=v&cmd=showvecinfo&vectorid=5495 Plasmid map source information]<br />
<br />
====Sequence====<br />
1 <br />
CACCTAAATT<br />
GTAAGCGTTA<br />
ATATTTTGTT<br />
AAAATTCGCG<br />
TTAAATTTTT<br />
GTTAAATCAG<br />
61 <br />
CTCATTTTTT<br />
AACCAATAGG<br />
CCGAAATCGG<br />
CAAAATCCCT<br />
TATAAATCAA<br />
AAGAATAGAC<br />
121 <br />
CGAGATAGGG<br />
TTGAGTGTTG<br />
TTCCAGTTTG<br />
GAACAAGAGT<br />
CCACTATTAA<br />
AGAACGTGGA<br />
181 <br />
CTCCAACGTC<br />
AAAGGGCGAA<br />
AAACCGTCTA<br />
TCAGGGCGAT<br />
GGCCCACTAC<br />
GTGAACCATC<br />
241 <br />
ACCCTAATCA<br />
AGTTTTTTGG<br />
GGTCGAGGTG<br />
CCGTAAAGCA<br />
CTAAATCGGA<br />
ACCCTAAAGG<br />
301 <br />
GAGCCCCCGA<br />
TTTAGAGCTT<br />
GACGGGGAAA<br />
GCCGGCGAAC<br />
GTGGCGAGAA<br />
AGGAAGGGAA<br />
361 <br />
GAAAGCGAAA<br />
GGAGCGGGCG<br />
CTAGGGCGCT<br />
GGCAAGTGTA<br />
GCGGTCACGC<br />
TGCGCGTAAC<br />
421 <br />
CACCACACCC<br />
GCCGCGCTTA<br />
ATGCGCCGCT<br />
ACAGGGCGCG<br />
TCCCATTCGC<br />
CATTCAGGCT<br />
481 <br />
GCGCAACTGT<br />
TGGGAAGGGC<br />
GATCGGTGCG<br />
GGCCTCTTCG<br />
CTATTACGCC<br />
AGCTGGCGAA<br />
541 <br />
AGGGGGATGT<br />
GCTGCAAGGC<br />
GATTAAGTTG<br />
GGTAACGCCA<br />
GGGTTTTCCC<br />
AGTCACGACG<br />
601 <br />
TTGTAAAACG<br />
ACGGCCAGTG<br />
AATTGTAATA<br />
CGACTCACTA<br />
TAGGGCGAAT<br />
TGGGTACCGG<br />
661 <br />
GCCCCCCCTC<br />
GAGGTCGACG<br />
GTATCGATAA<br />
GCTTGATATC<br />
GAATTCCTGC<br />
AGCCCGGGGG<br />
721 <br />
ATCCACTAGT<br />
TCTAGAGCGG<br />
CCGCCACCGC<br />
GGTGGAGCTC<br />
CAGCTTTTGT<br />
TCCCTTTAGT<br />
781 <br />
GAGGGTTAAT<br />
TTCGAGCTTG<br />
GCGTAATCAT<br />
GGTCATAGCT<br />
GTTTCCTGTG<br />
TGAAATTGTT<br />
841 <br />
ATCCGCTCAC<br />
AATTCCACAC<br />
AACATACGAG<br />
CCGGAAGCAT<br />
AAAGTGTAAA<br />
GCCTGGGGTG<br />
901 <br />
CCTAATGAGT<br />
GAGCTAACTC<br />
ACATTAATTG<br />
CGTTGCGCTC<br />
ACTGCCCGCT<br />
TTCCAGTCGG<br />
961 <br />
GAAACCTGTC<br />
GTGCCAGCTG<br />
CATTAATGAA<br />
TCGGCCAACG<br />
CGCGGGGAGA<br />
GGCGGTTTGC<br />
1021 <br />
GTATTGGGCG<br />
CTCTTCCGCT<br />
TCCTCGCTCA<br />
CTGACTCGCT<br />
GCGCTCGGTC<br />
GTTCGGCTGC<br />
1081 <br />
GGCGAGCGGT<br />
ATCAGCTCAC<br />
TCAAAGGCGG<br />
TAATACGGTT<br />
ATCCACAGAA<br />
TCAGGGGATA<br />
1141 <br />
ACGCAGGAAA<br />
GAACATGTGA<br />
GCAAAAGGCC<br />
AGCAAAAGGC<br />
CAGGAACCGT<br />
AAAAAGGCCG<br />
1201 <br />
CGTTGCTGGC<br />
GTTTTTCCAT<br />
AGGCTCCGCC<br />
CCCCTGACGA<br />
GCATCACAAA<br />
AATCGACGCT<br />
1261 <br />
CAAGTCAGAG<br />
GTGGCGAAAC<br />
CCGACAGGAC<br />
TATAAAGATA<br />
CCAGGCGTTT<br />
CCCCCTGGAA<br />
1321 <br />
GCTCCCTCGT<br />
GCGCTCTCCT<br />
GTTCCGACCC<br />
TGCCGCTTAC<br />
CGGATACCTG<br />
TCCGCCTTTC<br />
1381 <br />
TCCCTTCGGG<br />
AAGCGTGGCG<br />
CTTTCTCATA<br />
GCTCACGCTG<br />
TAGGTATCTC<br />
AGTTCGGTGT<br />
1441 <br />
AGGTCGTTCG<br />
CTCCAAGCTG<br />
GGCTGTGTGC<br />
ACGAACCCCC<br />
CGTTCAGCCC<br />
GACCGCTGCG<br />
1501 <br />
CCTTATCCGG<br />
TAACTATCGT<br />
CTTGAGTCCA<br />
ACCCGGTAAG<br />
ACACGACTTA<br />
TCGCCACTGG<br />
1561 <br />
CAGCAGCCAC<br />
TGGTAACAGG<br />
ATTAGCAGAG<br />
CGAGGTATGT<br />
AGGCGGTGCT<br />
ACAGAGTTCT<br />
1621 <br />
TGAAGTGGTG<br />
GCCTAACTAC<br />
GGCTACACTA<br />
GAAGGACAGT<br />
ATTTGGTATC<br />
TGCGCTCTGC<br />
1681 <br />
TGAAGCCAGT<br />
TACCTTCGGA<br />
AAAAGAGTTG<br />
GTAGCTCTTG<br />
ATCCGGCAAA<br />
CAAACCACCG<br />
1741 <br />
CTGGTAGCGG<br />
TGGTTTTTTT<br />
GTTTGCAAGC<br />
AGCAGATTAC<br />
GCGCAGAAAA<br />
AAAGGATCTC<br />
1801 <br />
AAGAAGATCC<br />
TTTGATCTTT<br />
TCTACGGGGT<br />
CTGACGCTCA<br />
GTGGAACGAA<br />
AACTCACGTT<br />
1861 <br />
AAGGGATTTT<br />
GGTCATGAGA<br />
TTATCAAAAA<br />
GGATCTTCAC<br />
CTAGATCCTT<br />
TTAAATTAAA<br />
1921 <br />
AATGAAGTTT<br />
TAAATCAATC<br />
TAAAGTATAT<br />
ATGAProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
AAAC<br />
TTGGTCTGAC<br />
AGTTACCAAT<br />
1981 <br />
GCTTAATCAG<br />
TGAGGCACCT<br />
ATCTCAGCGA<br />
TCTGTCTATT<br />
TCGTTCATCC<br />
ATAGTTGCCT<br />
2041 <br />
GACTCCCCGT<br />
CGTGTAGATA<br />
ACTACGATAC<br />
GGGAGGGCTT<br />
ACCATCTGGC<br />
CCCAGTGCTG<br />
2101 <br />
CAATGATACC<br />
GCGAGACCCA<br />
CGCTCACCGG<br />
CTCCAGATTT<br />
ATCAGCAATA<br />
AACCAGCCAG<br />
2161 <br />
CCGGAAGGGC<br />
CGAGCGCAGA<br />
AGTGGTCCTG<br />
CAACTTTATC<br />
CGCCTCCATC<br />
CAGTCTATTA<br />
2221 <br />
ATTGTTGCCG<br />
GGAAGCTAGA<br />
GTAAGTAGTT<br />
CGCCAGTTAA<br />
TAGTTTGCGC<br />
AACGTTGTTG<br />
2281 <br />
CCATTGCTAC<br />
AGGCATCGTG<br />
GTGTCACGCT<br />
CGTCGTTTGG<br />
TATGGCTTCA<br />
TTCAGCTCCG<br />
2341 <br />
GTTCCCAACG<br />
ATCAAGGCGA<br />
GTTACATGAT<br />
CCCCCATGTT<br />
GTGCAAAAAA<br />
GCGGTTAGCT<br />
2401 <br />
CCTTCGGTCC<br />
TCCGATCGTT<br />
GTCAGAAGTA<br />
AGTTGGCCGC<br />
AGTGTTATCA<br />
CTCATGGTTA<br />
2461 <br />
TGGCAGCACT<br />
GCATAATTCT<br />
CTTACTGTCA<br />
TGCCATCCGT<br />
AAGATGCTTT<br />
TCTGTGACTG<br />
2521 <br />
GTGAGTACTC<br />
AACCAAGTCA<br />
TTCTGAGAAT<br />
AGTGTATGCG<br />
GCGACCGAGT<br />
TGCTCTTGCC<br />
2581 <br />
CGGCGTCAAT<br />
ACGGGATAAT<br />
ACCGCGCCAC<br />
ATAGCAGAAC<br />
TTTAAAAGTG<br />
CTCATCATTG<br />
2641 <br />
GAAAACGTTC<br />
TTCGGGGCGA<br />
AAACTCTCAA<br />
GGATCTTACC<br />
GCTGTTGAGA<br />
TCCAGTTCGA<br />
2701 <br />
TGTAACCCAC<br />
TCGTGCACCC<br />
AACTGATCTT<br />
CAGCATCTTT<br />
TACTTTCACC<br />
AGCGTTTCTG<br />
2761 <br />
GGTGAGCAAA<br />
AACAGGAAGG<br />
CAAAATGCCG<br />
CAAAAAAGGG<br />
AATAAGGGCG<br />
ACACGGAAAT<br />
2821 <br />
GTTGAATACT<br />
CATACTCTTC<br />
CTTTTTCAAT<br />
ATTATTGAAG<br />
CATTTATCAG<br />
GGTTATTGTC</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/PlasmidsTeam:University of Chicago/Notebook/Plasmids2008-07-29T19:13:26Z<p>Norayucel: New page: ==pBluescript SK== [http://www.addgene.org/pgvec1?f=v&cmd=showvecinfo&vectorid=5495 Plasmid map source information]</p>
<hr />
<div>==pBluescript SK==<br />
[http://www.addgene.org/pgvec1?f=v&cmd=showvecinfo&vectorid=5495 Plasmid map source information]</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T19:09:17Z<p>Norayucel: /* Materials */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
* [[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]<br />
* [[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]<br />
<br />
Remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Materials ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
*[[Team:University_of_Chicago/Notebook/Plasmids|Plasmids]]<br />
<br />
== Protocols ==<br />
*[[Team:University_of_Chicago/Notebook/TOP10 competent cells|TOP10 competent cells]]<br />
*[[Team:University_of_Chicago/Notebook/Glycerol stocks|Glycerol stocks]]<br />
*[[Team:University_of_Chicago/Notebook/DNA mini-prep|DNA mini-prep]]</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T19:02:33Z<p>Norayucel: /* Recipes */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
* [[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]<br />
* [[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]<br />
<br />
Remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Materials ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
<br />
== Protocols ==<br />
*[[Team:University_of_Chicago/Notebook/TOP10 competent cells|TOP10 competent cells]]<br />
*[[Team:University_of_Chicago/Notebook/Glycerol stocks|Glycerol stocks]]<br />
*[[Team:University_of_Chicago/Notebook/DNA mini-prep|DNA mini-prep]]</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/DNA_mini-prepTeam:University of Chicago/Notebook/DNA mini-prep2008-07-29T18:56:04Z<p>Norayucel: /* Zyppy mini-prep */</p>
<hr />
<div>====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash<br />
<br />
<br />
# The Zyppy Elution Buffer cProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
tains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/DNA_mini-prepTeam:University of Chicago/Notebook/DNA mini-prep2008-07-29T18:55:23Z<p>Norayucel: /* Zyppy mini-prep */</p>
<hr />
<div>====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash BufferProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
<br />
# The ZyProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
py Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/DNA_mini-prepTeam:University of Chicago/Notebook/DNA mini-prep2008-07-29T18:54:45Z<p>Norayucel: /* Zyppy mini-prep */</p>
<hr />
<div>====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The ZyProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
y Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/DNA_mini-prepTeam:University of Chicago/Notebook/DNA mini-prep2008-07-29T18:54:13Z<p>Norayucel: New page: ====Zyppy mini-prep==== The following procedure is performed at room temperature. Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™...</p>
<hr />
<div>====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The Zyppy Elution BProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
fer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:53:05Z<p>Norayucel: /* DNA prep */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
* [[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]<br />
* [[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]<br />
<br />
Remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
<br />
== Protocols ==<br />
*[[Team:University_of_Chicago/Notebook/TOP10 competent cells|TOP10 competent cells]]<br />
*[[Team:University_of_Chicago/Notebook/Glycerol stocks|Glycerol stocks]]<br />
*[[Team:University_of_Chicago/Notebook/DNA mini-prep|DNA mini-prep]]</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/Glycerol_stocksTeam:University of Chicago/Notebook/Glycerol stocks2008-07-29T18:50:26Z<p>Norayucel: New page: ===== Glycerol Stocks ===== ====== Materials ====== * 40% glycerol solution * Cryogenic vials ====== Method ====== # Add 1 ml of 40% glycerol in H2O to a cryogenic vial. # Add 1 ml sa...</p>
<hr />
<div>===== Glycerol Stocks =====<br />
<br />
====== Materials ======<br />
* 40% glycerol solution <br />
* Cryogenic vials <br />
<br />
====== Method ======<br />
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial. <br />
# Add 1 ml sample from the culture of bacteria to be stored. <br />
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. <br />
#* Alternatively, pipet to mix. <br />
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. <br />
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. <br />
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. <br />
<br />
====== Notes ======<br />
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.<br />
Prof. Schonbaum's version<br />
# Grow a 3 ml culture overnight<br />
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols<br />
# Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:50:10Z<p>Norayucel: /* Protocols */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
* [[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]<br />
* [[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]<br />
* [[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]<br />
<br />
Remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
<br />
== Protocols ==<br />
*[[Team:University_of_Chicago/Notebook/TOP10 competent cells|TOP10 competent cells]]<br />
*[[Team:University_of_Chicago/Notebook/Glycerol stocks|Glycerol stocks]]<br />
*[[Team:University_of_Chicago/Notebook/DNA mini-prep|DNA mini-prep]]<br />
=== DNA prep ===<br />
<br />
====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete Proxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
utralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of tenProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
r less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The ZyppProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/TOP10_competent_cellsTeam:University of Chicago/Notebook/TOP10 competent cells2008-07-29T18:47:11Z<p>Norayucel: New page: ==== TOP10 Competent Cells ==== * Prechill plasticware and glassware ===== Preparing seed stocks ===== # Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C #* room ...</p>
<hr />
<div>==== TOP10 Competent Cells ====<br />
* Prechill plasticware and glassware<br />
<br />
===== Preparing seed stocks =====<br />
# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C <br />
#* room temperature works well <br />
# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C <br />
#* room temperature works well <br />
# Add glycerol to 15% <br />
# Aliquot 1 ml samples to Nunc cryotubes <br />
# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes <br />
#* This step may not be necessary <br />
# Place in -80°C freezer indefinitely.<br />
<br />
===== Preparing competent cells =====<br />
# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 <br />
#* This takes approximately 16 hours. <br />
#* Controlling the temperature makes this a more reproducible process, but is not essential. <br />
#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule <br />
#* Aim for lower, not higher OD if you can't hit this mark <br />
# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle. <br />
#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend <br />
#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer <br />
# Gently resuspend in 80 ml of ice cold CCMB80 buffer <br />
#* sometimes this is less than completely gentle. It still works. <br />
# Incubate on ice 20 minutes <br />
# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer. <br />
# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells. <br />
# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. <br />
# Incubate on ice for 20 minutes <br />
# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates <br />
# Store at -80°C indefinitely. <br />
#* Flash freezing does not appear to be necessary <br />
# Test competence (see below) <br />
# Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. <br />
<br />
===== Measurement of competence =====<br />
# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) <br />
#* This is at 10 pg/_l or 10-5 _g/_l <br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours <br />
# Heat shock 60 sec at 42C <br />
# Add 250 _l SOC <br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated <br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. <br />
#* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicoProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
and tetracycline resistant, we find growing for 2 hours yields many more colonies <br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads <br />
#* Good cells should yield around 100 - 400 colonies <br />
#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA <br />
#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:46:46Z<p>Norayucel: /* Protocols */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
To see what each of us has done each day, go to [[Team:University_of_Chicago/Notebook/Individual notebook pages|individual notebook pages]]. And remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
<br />
== Protocols ==<br />
*[[Team:University_of_Chicago/Notebook/TOP10 competent cells|TOP10 competent cells]]<br />
<br />
===== Glycerol Stocks =====<br />
<br />
====== Materials ======<br />
* 40% glycerol solution <br />
* Cryogenic vials <br />
<br />
====== Method ======<br />
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial. <br />
# Add 1 ml sample from the culture of bacteria to be stored. <br />
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. <br />
#* Alternatively, pipet to mix. <br />
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. <br />
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. <br />
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. <br />
<br />
====== Notes ======<br />
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.<br />
Prof. Schonbaum's version<br />
# Grow a 3 ml culture overnight<br />
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols<br />
# Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.<br />
<br />
=== DNA prep ===<br />
<br />
====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete Proxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
utralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The ZyppProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:44:18Z<p>Norayucel: /* Recipes */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
To see what each of us has done each day, go to [[Team:University_of_Chicago/Notebook/Individual notebook pages|individual notebook pages]]. And remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
<br />
== Protocols ==<br />
=== Growing Cells ===<br />
==== TOP10 Competent Cells ====<br />
* Prechill plasticware and glassware<br />
<br />
===== Preparing seed stocks =====<br />
# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C <br />
#* room temperature works well <br />
# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C <br />
#* room temperature works well <br />
# Add glycerol to 15% <br />
# Aliquot 1 ml samples to Nunc cryotubes <br />
# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes <br />
#* This step may not be necessary <br />
# Place in -80°C freezer indefinitely.<br />
<br />
===== Preparing competent cells =====<br />
# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 <br />
#* This takes approximately 16 hours. <br />
#* Controlling the temperature makes this a more reproducible process, but is not essential. <br />
#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule <br />
#* Aim for lower, not higher OD if you can't hit this mark <br />
# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle. <br />
#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend <br />
#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer <br />
# Gently resuspend in 80 ml of ice cold CCMB80 buffer <br />
#* sometimes this is less than completely gentle. It still works. <br />
# Incubate on ice 20 minutes <br />
# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer. <br />
# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells. <br />
# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. <br />
# Incubate on ice for 20 minutes <br />
# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates <br />
# Store at -80°C indefinitely. <br />
#* Flash freezing does not appear to be necessary <br />
# Test competence (see below) <br />
# Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. <br />
<br />
===== Measurement of competence =====<br />
# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) <br />
#* This is at 10 pg/_l or 10-5 _g/_l <br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours <br />
# Heat shock 60 sec at 42C <br />
# Add 250 _l SOC <br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated <br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. <br />
#* For our plasmids (pSB1AC3, pSProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
AT3) which are chloramphProxy-CProxyProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
onneProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
ion: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
nection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
icol anProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
tetracycline resistant, we find growing for 2 hours yields many more colonies <br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads <br />
#* Good cells should yield around 100 - 400 colonies <br />
#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA <br />
#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA <br />
<br />
===== Glycerol Stocks =====<br />
<br />
====== Materials ======<br />
* 40% glycerol solution <br />
* Cryogenic vials <br />
<br />
====== Method ======<br />
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial. <br />
# Add 1 ml sample from the culture of bacteria to be stored. <br />
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. <br />
#* Alternatively, pipet to mix. <br />
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. <br />
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. <br />
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. <br />
<br />
====== Notes ======<br />
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.<br />
Prof. Schonbaum's version<br />
# Grow a 3 ml culture overnight<br />
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols<br />
# Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.<br />
<br />
=== DNA prep ===<br />
<br />
====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The ZyppProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/GelsTeam:University of Chicago/Notebook/Gels2008-07-29T18:43:47Z<p>Norayucel: /* 1.2% */</p>
<hr />
<div>=== DNA and protein gels ===<br />
==== Agar ====<br />
===== 0.8% =====<br />
# Weigh out .8g agarose<br />
# Put agarose in 500mL Erlenmeyer flask.<br />
# Add 100mL 1X TBE Buffer. Swirl.<br />
# Microwave at 100% power until the agarose starts to dissolve. Every 30-40 seconds, stop and swirl. Continue microwaving until solution boils and agarose is dissolved.<br />
# After agarose is dissolved, cover flask with foil or aran wrap and place in 55-60C waterbath until needed. <br />
# When Agarose has cooled to enough to touch comfortably, add 10microliters Syber Safe. Swirl.<br />
# Pour 30mL gels. Use 50Ml conical tube to measure 30mL.<br />
===== 1.2% =====<br />
Same as above but use 1.2 g agarose instead of .8g<br />
<br />
====Coomassie Blue Stain/Destain Recipe====<br />
<br />
450 ml water<br><br />
450 ml methanol<br><br />
100 ml 100% (glacial) acetic acid<br><br><br />
<br />
#Add 2 g per liter coomassie blue for stain. Destain is exactly the same, except do not add coomassie blue.<br />
#To stain the gel, incubate it with gentle shaking for at least 30 minutes. Pour the stain back into the bottle (you can reuse it). Rinse out the residual stain with a small amount of destain solution, and then incubate with gentle shaking for ~1 hour or more. To speed up destaining, you can put some folded KimWipes in the dish. The dye will adsorb to these, which will remove it from solution.<br />
#After your gel is destained, you can rehydrate it in distilled water and then image it and/or place it on a piece of filter paper and dry it with a gel dryer.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/GelsTeam:University of Chicago/Notebook/Gels2008-07-29T18:43:33Z<p>Norayucel: New page: === DNA and protein gels === ==== Agar ==== ===== 0.8% ===== # Weigh out .8g agarose # Put agarose in 500mL Erlenmeyer flask. # Add 100mL 1X TBE Buffer. Swirl. # Microwave at 100% power un...</p>
<hr />
<div>=== DNA and protein gels ===<br />
==== Agar ====<br />
===== 0.8% =====<br />
# Weigh out .8g agarose<br />
# Put agarose in 500mL Erlenmeyer flask.<br />
# Add 100mL 1X TBE Buffer. Swirl.<br />
# Microwave at 100% power until the agarose starts to dissolve. Every 30-40 seconds, stop and swirl. Continue microwaving until solution boils and agarose is dissolved.<br />
# After agarose is dissolved, cover flask with foil or aran wrap and place in 55-60C waterbath until needed. <br />
# When Agarose has cooled to enough to touch comfortably, add 10microliters Syber Safe. Swirl.<br />
# Pour 30mL gels. Use 50Ml conical tube to measure 30mL.<br />
===== 1.2% =====<br />
Same as above but use 1.2 g agarose instead of .8g<br />
<br />
<br />
====Coomassie Blue Stain/Destain Recipe====<br />
<br />
450 ml water<br><br />
450 ml methanol<br><br />
100 ml 100% (glacial) acetic acid<br><br><br />
<br />
#Add 2 g per liter coomassie blue for stain. Destain is exactly the same, except do not add coomassie blue.<br />
#To stain the gel, incubate it with gentle shaking for at least 30 minutes. Pour the stain back into the bottle (you can reuse it). Rinse out the residual stain with a small amount of destain solution, and then incubate with gentle shaking for ~1 hour or more. To speed up destaining, you can put some folded KimWipes in the dish. The dye will adsorb to these, which will remove it from solution.<br />
#After your gel is destained, you can rehydrate it in distilled water and then image it and/or place it on a piece of filter paper and dry it with a gel dryer.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:43:24Z<p>Norayucel: /* DNA and protein gels */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
To see what each of us has done each day, go to [[Team:University_of_Chicago/Notebook/Individual notebook pages|individual notebook pages]]. And remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
<br />
<br />
<br />
<br />
== Protocols ==<br />
=== Growing Cells ===<br />
==== TOP10 Competent Cells ====<br />
* Prechill plasticware and glassware<br />
<br />
===== Preparing seed stocks =====<br />
# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C <br />
#* room temperature works well <br />
# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C <br />
#* room temperature works well <br />
# Add glycerol to 15% <br />
# Aliquot 1 ml samples to Nunc cryotubes <br />
# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes <br />
#* This step may not be necessary <br />
# Place in -80°C freezer indefinitely.<br />
<br />
===== Preparing competent cells =====<br />
# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 <br />
#* This takes approximately 16 hours. <br />
#* Controlling the temperature makes this a more reproducible process, but is not essential. <br />
#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule <br />
#* Aim for lower, not higher OD if you can't hit this mark <br />
# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle. <br />
#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend <br />
#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer <br />
# Gently resuspend in 80 ml of ice cold CCMB80 buffer <br />
#* sometimes this is less than completely gentle. It still works. <br />
# Incubate on ice 20 minutes <br />
# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer. <br />
# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells. <br />
# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. <br />
# Incubate on ice for 20 minutes <br />
# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates <br />
# Store at -80°C indefinitely. <br />
#* Flash freezing does not appear to be necessary <br />
# Test competence (see below) <br />
# Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. <br />
<br />
===== Measurement of competence =====<br />
# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) <br />
#* This is at 10 pg/_l or 10-5 _g/_l <br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours <br />
# Heat shock 60 sec at 42C <br />
# Add 250 _l SOC <br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated <br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. <br />
#* For our plasmids (pSB1AC3, pSProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
AT3) which are chloramphProxy-CProxyProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
onneProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
ion: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
nection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
icol anProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
tetracycline resistant, we find growing for 2 hours yields many more colonies <br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads <br />
#* Good cells should yield around 100 - 400 colonies <br />
#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA <br />
#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA <br />
<br />
===== Glycerol Stocks =====<br />
<br />
====== Materials ======<br />
* 40% glycerol solution <br />
* Cryogenic vials <br />
<br />
====== Method ======<br />
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial. <br />
# Add 1 ml sample from the culture of bacteria to be stored. <br />
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. <br />
#* Alternatively, pipet to mix. <br />
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. <br />
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. <br />
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. <br />
<br />
====== Notes ======<br />
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.<br />
Prof. Schonbaum's version<br />
# Grow a 3 ml culture overnight<br />
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols<br />
# Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.<br />
<br />
=== DNA prep ===<br />
<br />
====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The ZyppProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:43:00Z<p>Norayucel: /* Recipes */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
To see what each of us has done each day, go to [[Team:University_of_Chicago/Notebook/Individual notebook pages|individual notebook pages]]. And remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]<br />
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]<br />
<br />
<br />
=== DNA and protein gels ===<br />
==== Agar ====<br />
===== 0.8% =====<br />
# Weigh out .8g agarose<br />
# Put agarose in 500mL Erlenmeyer flask.<br />
# Add 100mL 1X TBE Buffer. Swirl.<br />
# Microwave at 100% power until the agarose starts to dissolve. Every 30-40 seconds, stop and swirl. Continue microwaving until solution boils and agarose is dissolved.<br />
# After agarose is dissolved, cover flask with foil or aran wrap and place in 55-60C waterbath until needed. <br />
# When Agarose has cooled to enough to touch comfortably, add 10microliters Syber Safe. Swirl.<br />
# Pour 30mL gels. Use 50Ml conical tube to measure 30mL.<br />
===== 1.2% =====<br />
Same as above but use 1.2 g agarose instead of .8g<br />
<br />
<br />
====Coomassie Blue Stain/Destain Recipe====<br />
<br />
450 ml water<br><br />
450 ml methanol<br><br />
100 ml 100% (glacial) acetic acid<br><br><br />
<br />
#Add 2 g per liter coomassie blue for stain. Destain is exactly the same, except do not add coomassie blue.<br />
#To stain the gel, incubate it with gentle shaking for at least 30 minutes. Pour the stain back into the bottle (you can reuse it). Rinse out the residual stain with a small amount of destain solution, and then incubate with gentle shaking for ~1 hour or more. To speed up destaining, you can put some folded KimWipes in the dish. The dye will adsorb to these, which will remove it from solution.<br />
#After your gel is destained, you can rehydrate it in distilled water and then image it and/or place it on a piece of filter paper and dry it with a gel dryer.<br />
<br />
== Protocols ==<br />
=== Growing Cells ===<br />
==== TOP10 Competent Cells ====<br />
* Prechill plasticware and glassware<br />
<br />
===== Preparing seed stocks =====<br />
# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C <br />
#* room temperature works well <br />
# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C <br />
#* room temperature works well <br />
# Add glycerol to 15% <br />
# Aliquot 1 ml samples to Nunc cryotubes <br />
# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes <br />
#* This step may not be necessary <br />
# Place in -80°C freezer indefinitely.<br />
<br />
===== Preparing competent cells =====<br />
# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 <br />
#* This takes approximately 16 hours. <br />
#* Controlling the temperature makes this a more reproducible process, but is not essential. <br />
#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule <br />
#* Aim for lower, not higher OD if you can't hit this mark <br />
# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle. <br />
#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend <br />
#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer <br />
# Gently resuspend in 80 ml of ice cold CCMB80 buffer <br />
#* sometimes this is less than completely gentle. It still works. <br />
# Incubate on ice 20 minutes <br />
# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer. <br />
# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells. <br />
# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. <br />
# Incubate on ice for 20 minutes <br />
# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates <br />
# Store at -80°C indefinitely. <br />
#* Flash freezing does not appear to be necessary <br />
# Test competence (see below) <br />
# Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. <br />
<br />
===== Measurement of competence =====<br />
# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) <br />
#* This is at 10 pg/_l or 10-5 _g/_l <br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours <br />
# Heat shock 60 sec at 42C <br />
# Add 250 _l SOC <br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated <br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. <br />
#* For our plasmids (pSB1AC3, pSProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
AT3) which are chloramphProxy-CProxyProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
onneProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
ion: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
nection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
icol anProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
tetracycline resistant, we find growing for 2 hours yields many more colonies <br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads <br />
#* Good cells should yield around 100 - 400 colonies <br />
#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA <br />
#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA <br />
<br />
===== Glycerol Stocks =====<br />
<br />
====== Materials ======<br />
* 40% glycerol solution <br />
* Cryogenic vials <br />
<br />
====== Method ======<br />
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial. <br />
# Add 1 ml sample from the culture of bacteria to be stored. <br />
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. <br />
#* Alternatively, pipet to mix. <br />
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. <br />
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. <br />
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. <br />
<br />
====== Notes ======<br />
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.<br />
Prof. Schonbaum's version<br />
# Grow a 3 ml culture overnight<br />
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols<br />
# Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.<br />
<br />
=== DNA prep ===<br />
<br />
====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The ZyppProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:41:48Z<p>Norayucel: /* Media */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
To see what each of us has done each day, go to [[Team:University_of_Chicago/Notebook/Individual notebook pages|individual notebook pages]]. And remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
*[[Team:University_of_Chicago/Notebook/Media|Media]]<br />
<br />
==== [[Team:University_of_Chicago/Notebook/Buffers|Buffers]] ====<br />
<br />
<br />
<br />
=== DNA and protein gels ===<br />
==== Agar ====<br />
===== 0.8% =====<br />
# Weigh out .8g agarose<br />
# Put agarose in 500mL Erlenmeyer flask.<br />
# Add 100mL 1X TBE Buffer. Swirl.<br />
# Microwave at 100% power until the agarose starts to dissolve. Every 30-40 seconds, stop and swirl. Continue microwaving until solution boils and agarose is dissolved.<br />
# After agarose is dissolved, cover flask with foil or aran wrap and place in 55-60C waterbath until needed. <br />
# When Agarose has cooled to enough to touch comfortably, add 10microliters Syber Safe. Swirl.<br />
# Pour 30mL gels. Use 50Ml conical tube to measure 30mL.<br />
===== 1.2% =====<br />
Same as above but use 1.2 g agarose instead of .8g<br />
<br />
<br />
====Coomassie Blue Stain/Destain Recipe====<br />
<br />
450 ml water<br><br />
450 ml methanol<br><br />
100 ml 100% (glacial) acetic acid<br><br><br />
<br />
#Add 2 g per liter coomassie blue for stain. Destain is exactly the same, except do not add coomassie blue.<br />
#To stain the gel, incubate it with gentle shaking for at least 30 minutes. Pour the stain back into the bottle (you can reuse it). Rinse out the residual stain with a small amount of destain solution, and then incubate with gentle shaking for ~1 hour or more. To speed up destaining, you can put some folded KimWipes in the dish. The dye will adsorb to these, which will remove it from solution.<br />
#After your gel is destained, you can rehydrate it in distilled water and then image it and/or place it on a piece of filter paper and dry it with a gel dryer.<br />
<br />
== Protocols ==<br />
=== Growing Cells ===<br />
==== TOP10 Competent Cells ====<br />
* Prechill plasticware and glassware<br />
<br />
===== Preparing seed stocks =====<br />
# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C <br />
#* room temperature works well <br />
# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C <br />
#* room temperature works well <br />
# Add glycerol to 15% <br />
# Aliquot 1 ml samples to Nunc cryotubes <br />
# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes <br />
#* This step may not be necessary <br />
# Place in -80°C freezer indefinitely.<br />
<br />
===== Preparing competent cells =====<br />
# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 <br />
#* This takes approximately 16 hours. <br />
#* Controlling the temperature makes this a more reproducible process, but is not essential. <br />
#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule <br />
#* Aim for lower, not higher OD if you can't hit this mark <br />
# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle. <br />
#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend <br />
#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer <br />
# Gently resuspend in 80 ml of ice cold CCMB80 buffer <br />
#* sometimes this is less than completely gentle. It still works. <br />
# Incubate on ice 20 minutes <br />
# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer. <br />
# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells. <br />
# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. <br />
# Incubate on ice for 20 minutes <br />
# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates <br />
# Store at -80°C indefinitely. <br />
#* Flash freezing does not appear to be necessary <br />
# Test competence (see below) <br />
# Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. <br />
<br />
===== Measurement of competence =====<br />
# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) <br />
#* This is at 10 pg/_l or 10-5 _g/_l <br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours <br />
# Heat shock 60 sec at 42C <br />
# Add 250 _l SOC <br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated <br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. <br />
#* For our plasmids (pSB1AC3, pSProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
AT3) which are chloramphProxy-CProxyProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
onneProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
ion: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
nection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
icol anProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
tetracycline resistant, we find growing for 2 hours yields many more colonies <br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads <br />
#* Good cells should yield around 100 - 400 colonies <br />
#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA <br />
#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA <br />
<br />
===== Glycerol Stocks =====<br />
<br />
====== Materials ======<br />
* 40% glycerol solution <br />
* Cryogenic vials <br />
<br />
====== Method ======<br />
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial. <br />
# Add 1 ml sample from the culture of bacteria to be stored. <br />
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. <br />
#* Alternatively, pipet to mix. <br />
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. <br />
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. <br />
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. <br />
<br />
====== Notes ======<br />
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.<br />
Prof. Schonbaum's version<br />
# Grow a 3 ml culture overnight<br />
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols<br />
# Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.<br />
<br />
=== DNA prep ===<br />
<br />
====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The ZyppProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:41:29Z<p>Norayucel: /* Buffers */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
To see what each of us has done each day, go to [[Team:University_of_Chicago/Notebook/Individual notebook pages|individual notebook pages]]. And remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
==== [[Team:University_of_Chicago/Notebook/Media|Media]] ====<br />
==== [[Team:University_of_Chicago/Notebook/Buffers|Buffers]] ====<br />
<br />
<br />
<br />
=== DNA and protein gels ===<br />
==== Agar ====<br />
===== 0.8% =====<br />
# Weigh out .8g agarose<br />
# Put agarose in 500mL Erlenmeyer flask.<br />
# Add 100mL 1X TBE Buffer. Swirl.<br />
# Microwave at 100% power until the agarose starts to dissolve. Every 30-40 seconds, stop and swirl. Continue microwaving until solution boils and agarose is dissolved.<br />
# After agarose is dissolved, cover flask with foil or aran wrap and place in 55-60C waterbath until needed. <br />
# When Agarose has cooled to enough to touch comfortably, add 10microliters Syber Safe. Swirl.<br />
# Pour 30mL gels. Use 50Ml conical tube to measure 30mL.<br />
===== 1.2% =====<br />
Same as above but use 1.2 g agarose instead of .8g<br />
<br />
<br />
====Coomassie Blue Stain/Destain Recipe====<br />
<br />
450 ml water<br><br />
450 ml methanol<br><br />
100 ml 100% (glacial) acetic acid<br><br><br />
<br />
#Add 2 g per liter coomassie blue for stain. Destain is exactly the same, except do not add coomassie blue.<br />
#To stain the gel, incubate it with gentle shaking for at least 30 minutes. Pour the stain back into the bottle (you can reuse it). Rinse out the residual stain with a small amount of destain solution, and then incubate with gentle shaking for ~1 hour or more. To speed up destaining, you can put some folded KimWipes in the dish. The dye will adsorb to these, which will remove it from solution.<br />
#After your gel is destained, you can rehydrate it in distilled water and then image it and/or place it on a piece of filter paper and dry it with a gel dryer.<br />
<br />
== Protocols ==<br />
=== Growing Cells ===<br />
==== TOP10 Competent Cells ====<br />
* Prechill plasticware and glassware<br />
<br />
===== Preparing seed stocks =====<br />
# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C <br />
#* room temperature works well <br />
# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C <br />
#* room temperature works well <br />
# Add glycerol to 15% <br />
# Aliquot 1 ml samples to Nunc cryotubes <br />
# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes <br />
#* This step may not be necessary <br />
# Place in -80°C freezer indefinitely.<br />
<br />
===== Preparing competent cells =====<br />
# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 <br />
#* This takes approximately 16 hours. <br />
#* Controlling the temperature makes this a more reproducible process, but is not essential. <br />
#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule <br />
#* Aim for lower, not higher OD if you can't hit this mark <br />
# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle. <br />
#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend <br />
#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer <br />
# Gently resuspend in 80 ml of ice cold CCMB80 buffer <br />
#* sometimes this is less than completely gentle. It still works. <br />
# Incubate on ice 20 minutes <br />
# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer. <br />
# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells. <br />
# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. <br />
# Incubate on ice for 20 minutes <br />
# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates <br />
# Store at -80°C indefinitely. <br />
#* Flash freezing does not appear to be necessary <br />
# Test competence (see below) <br />
# Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. <br />
<br />
===== Measurement of competence =====<br />
# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) <br />
#* This is at 10 pg/_l or 10-5 _g/_l <br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours <br />
# Heat shock 60 sec at 42C <br />
# Add 250 _l SOC <br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated <br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. <br />
#* For our plasmids (pSB1AC3, pSProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
AT3) which are chloramphProxy-CProxyProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
onneProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
ion: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
nection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
icol anProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
tetracycline resistant, we find growing for 2 hours yields many more colonies <br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads <br />
#* Good cells should yield around 100 - 400 colonies <br />
#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA <br />
#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA <br />
<br />
===== Glycerol Stocks =====<br />
<br />
====== Materials ======<br />
* 40% glycerol solution <br />
* Cryogenic vials <br />
<br />
====== Method ======<br />
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial. <br />
# Add 1 ml sample from the culture of bacteria to be stored. <br />
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. <br />
#* Alternatively, pipet to mix. <br />
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. <br />
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. <br />
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. <br />
<br />
====== Notes ======<br />
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.<br />
Prof. Schonbaum's version<br />
# Grow a 3 ml culture overnight<br />
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols<br />
# Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.<br />
<br />
=== DNA prep ===<br />
<br />
====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The ZyppProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/Notebook/BuffersTeam:University of Chicago/Notebook/Buffers2008-07-29T18:40:39Z<p>Norayucel: New page: === Buffers === ==== CCMB80 ==== * 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L) * 80 mM CaCl2.2H2O (11.8 g/L) * 20 mM MnCl2.4H2O (4.0 g/L) * 10 mM MgCl2.6H2O (2.0 g/L) * 10% glycerol (10...</p>
<hr />
<div>=== Buffers ===<br />
==== CCMB80 ==== <br />
* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L) <br />
* 80 mM CaCl2.2H2O (11.8 g/L) <br />
* 20 mM MnCl2.4H2O (4.0 g/L) <br />
* 10 mM MgCl2.6H2O (2.0 g/L) <br />
* 10% glycerol (100 ml/L) <br />
* adjust pH DOWN to 6.4 with 0.1N HCl if necessary <br />
** adjusting pH up will precipitate manganese dioxide from Mn containing solutions. <br />
* sterile filter and store at 4°C <br />
* slight dark precipitate appears not to affect its function<br />
<br />
*Note: Create 1M concentration stocks to make buffer prep easier in future.<br />
'''CCMB80: with aqueous stocks'''<br />
*10 mM KOAc pH 7.0: 10mL of 1M stock/L<br />
*80 mM CaCl2.2H2O: 80mL/L<br />
*20 mM MnCl2.4H2O: 20mL/L<br />
*10 mM MgCl2.6H2O: 10mL/L<br />
*10% glycerol: 100 ml/L<br />
<br />
==== Hutner's Mineral Base Concentrate ====<br />
It is not difficult, but follow directions carefully.<br />
<br />
* 20g nitrilotriacetic acid (NTA)<br />
* 54.5g magnesium sulfate heptahydrate<br />
* 6.67g calcium chloride dihydrate<br />
* 18.5mg ammonium molybdate tetrahydrate<br />
* 198mg iron sulfate monohydrate (or 323.7mg of the heptahydrate)<br />
* 100mL Metals-44<br />
<br />
TO BE CONTINUED<br />
<br />
==== 5x Ligation Adjustment Buffer ====<br />
* Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer <br />
* KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0)<br />
* CaCl2 400 mM (200 ml/l of a 2 M solution) <br />
* MnCl2 100 mM (100 ml/l of a 1 M solution) <br />
* Glycerol 46.8% (468 ml/liter) <br />
* pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter) <br />
** Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --Meaganl 15:50, 25 January 2007 (EST) <br />
* water to 1 liter <br />
* autoclave or sterile filter <br />
* Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5 <br />
* Reshma 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:39:58Z<p>Norayucel: /* Media */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
To see what each of us has done each day, go to [[Team:University_of_Chicago/Notebook/Individual notebook pages|individual notebook pages]]. And remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
==== [[Team:University_of_Chicago/Notebook/Media|Media]] ====<br />
==== [[Team:University_of_Chicago/Notebook/Buffers|Buffers]] ====<br />
<br />
=== Buffers ===<br />
==== CCMB80 ==== <br />
* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L) <br />
* 80 mM CaCl2.2H2O (11.8 g/L) <br />
* 20 mM MnCl2.4H2O (4.0 g/L) <br />
* 10 mM MgCl2.6H2O (2.0 g/L) <br />
* 10% glycerol (100 ml/L) <br />
* adjust pH DOWN to 6.4 with 0.1N HCl if necessary <br />
** adjusting pH up will precipitate manganese dioxide from Mn containing solutions. <br />
* sterile filter and store at 4°C <br />
* slight dark precipitate appears not to affect its function<br />
<br />
*Note: Create 1M concentration stocks to make buffer prep easier in future.<br />
'''CCMB80: with aqueous stocks'''<br />
*10 mM KOAc pH 7.0: 10mL of 1M stock/L<br />
*80 mM CaCl2.2H2O: 80mL/L<br />
*20 mM MnCl2.4H2O: 20mL/L<br />
*10 mM MgCl2.6H2O: 10mL/L<br />
*10% glycerol: 100 ml/L<br />
<br />
==== Hutner's Mineral Base Concentrate ====<br />
It is not difficult, but follow directions carefully.<br />
<br />
* 20g nitrilotriacetic acid (NTA)<br />
* 54.5g magnesium sulfate heptahydrate<br />
* 6.67g calcium chloride dihydrate<br />
* 18.5mg ammonium molybdate tetrahydrate<br />
* 198mg iron sulfate monohydrate (or 323.7mg of the heptahydrate)<br />
* 100mL Metals-44<br />
<br />
TO BE CONTINUED<br />
<br />
==== 5x Ligation Adjustment Buffer ====<br />
* Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer <br />
* KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0)<br />
* CaCl2 400 mM (200 ml/l of a 2 M solution) <br />
* MnCl2 100 mM (100 ml/l of a 1 M solution) <br />
* Glycerol 46.8% (468 ml/liter) <br />
* pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter) <br />
** Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --Meaganl 15:50, 25 January 2007 (EST) <br />
* water to 1 liter <br />
* autoclave or sterile filter <br />
* Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5 <br />
* Reshma 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional.<br />
<br />
=== DNA and protein gels ===<br />
==== Agar ====<br />
===== 0.8% =====<br />
# Weigh out .8g agarose<br />
# Put agarose in 500mL Erlenmeyer flask.<br />
# Add 100mL 1X TBE Buffer. Swirl.<br />
# Microwave at 100% power until the agarose starts to dissolve. Every 30-40 seconds, stop and swirl. Continue microwaving until solution boils and agarose is dissolved.<br />
# After agarose is dissolved, cover flask with foil or aran wrap and place in 55-60C waterbath until needed. <br />
# When Agarose has cooled to enough to touch comfortably, add 10microliters Syber Safe. Swirl.<br />
# Pour 30mL gels. Use 50Ml conical tube to measure 30mL.<br />
===== 1.2% =====<br />
Same as above but use 1.2 g agarose instead of .8g<br />
<br />
<br />
====Coomassie Blue Stain/Destain Recipe====<br />
<br />
450 ml water<br><br />
450 ml methanol<br><br />
100 ml 100% (glacial) acetic acid<br><br><br />
<br />
#Add 2 g per liter coomassie blue for stain. Destain is exactly the same, except do not add coomassie blue.<br />
#To stain the gel, incubate it with gentle shaking for at least 30 minutes. Pour the stain back into the bottle (you can reuse it). Rinse out the residual stain with a small amount of destain solution, and then incubate with gentle shaking for ~1 hour or more. To speed up destaining, you can put some folded KimWipes in the dish. The dye will adsorb to these, which will remove it from solution.<br />
#After your gel is destained, you can rehydrate it in distilled water and then image it and/or place it on a piece of filter paper and dry it with a gel dryer.<br />
<br />
== Protocols ==<br />
=== Growing Cells ===<br />
==== TOP10 Competent Cells ====<br />
* Prechill plasticware and glassware<br />
<br />
===== Preparing seed stocks =====<br />
# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C <br />
#* room temperature works well <br />
# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C <br />
#* room temperature works well <br />
# Add glycerol to 15% <br />
# Aliquot 1 ml samples to Nunc cryotubes <br />
# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes <br />
#* This step may not be necessary <br />
# Place in -80°C freezer indefinitely.<br />
<br />
===== Preparing competent cells =====<br />
# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 <br />
#* This takes approximately 16 hours. <br />
#* Controlling the temperature makes this a more reproducible process, but is not essential. <br />
#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule <br />
#* Aim for lower, not higher OD if you can't hit this mark <br />
# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle. <br />
#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend <br />
#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer <br />
# Gently resuspend in 80 ml of ice cold CCMB80 buffer <br />
#* sometimes this is less than completely gentle. It still works. <br />
# Incubate on ice 20 minutes <br />
# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer. <br />
# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells. <br />
# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. <br />
# Incubate on ice for 20 minutes <br />
# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates <br />
# Store at -80°C indefinitely. <br />
#* Flash freezing does not appear to be necessary <br />
# Test competence (see below) <br />
# Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. <br />
<br />
===== Measurement of competence =====<br />
# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) <br />
#* This is at 10 pg/_l or 10-5 _g/_l <br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours <br />
# Heat shock 60 sec at 42C <br />
# Add 250 _l SOC <br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated <br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. <br />
#* For our plasmids (pSB1AC3, pSProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
AT3) which are chloramphProxy-CProxyProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
onneProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
ion: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
nection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
icol anProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
tetracycline resistant, we find growing for 2 hours yields many more colonies <br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads <br />
#* Good cells should yield around 100 - 400 colonies <br />
#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA <br />
#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA <br />
<br />
===== Glycerol Stocks =====<br />
<br />
====== Materials ======<br />
* 40% glycerol solution <br />
* Cryogenic vials <br />
<br />
====== Method ======<br />
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial. <br />
# Add 1 ml sample from the culture of bacteria to be stored. <br />
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. <br />
#* Alternatively, pipet to mix. <br />
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. <br />
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. <br />
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. <br />
<br />
====== Notes ======<br />
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.<br />
Prof. Schonbaum's version<br />
# Grow a 3 ml culture overnight<br />
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols<br />
# Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.<br />
<br />
=== DNA prep ===<br />
<br />
====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
# The ZyppProxy-Connection: keep-alive<br />
Cache-Control: max-age=0<br />
<br />
�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
pH) can also be used to elute the DNA.</div>Norayucelhttp://2008.igem.org/Team:University_of_Chicago/NotebookTeam:University of Chicago/Notebook2008-07-29T18:39:26Z<p>Norayucel: /* Media */</p>
<hr />
<div>{{Masthead}}<br />
<br />
<br />
==Lab Calendar==<br />
<br />
Click to see what lab work was done each day!<br><br><br />
<div align="center"><br />
<html><br />
<iframe src="http://www.google.com/calendar/embed?src=rn71lt2mhnqu0neu4ini1hi91s%40group.calendar.google.com&ctz=America/Chicago" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe> <br />
</html></div><br />
<br><br />
==Individual Lab Notebook Pages==<br />
<br />
To see what each of us has done each day, go to [[Team:University_of_Chicago/Notebook/Individual notebook pages|individual notebook pages]]. And remember: '''If you did it, record it'''<br><br />
<br />
== Resources ==<br />
<br />
[http://www.freewebs.com/genehackers/ Team Website (External Link)]<br />
<br />
== Recipes ==<br />
==== [[Team:University_of_Chicago/Notebook/Media|Media]] ====<br />
<br />
<br />
<br />
=== Buffers ===<br />
==== CCMB80 ==== <br />
* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L) <br />
* 80 mM CaCl2.2H2O (11.8 g/L) <br />
* 20 mM MnCl2.4H2O (4.0 g/L) <br />
* 10 mM MgCl2.6H2O (2.0 g/L) <br />
* 10% glycerol (100 ml/L) <br />
* adjust pH DOWN to 6.4 with 0.1N HCl if necessary <br />
** adjusting pH up will precipitate manganese dioxide from Mn containing solutions. <br />
* sterile filter and store at 4°C <br />
* slight dark precipitate appears not to affect its function<br />
<br />
*Note: Create 1M concentration stocks to make buffer prep easier in future.<br />
'''CCMB80: with aqueous stocks'''<br />
*10 mM KOAc pH 7.0: 10mL of 1M stock/L<br />
*80 mM CaCl2.2H2O: 80mL/L<br />
*20 mM MnCl2.4H2O: 20mL/L<br />
*10 mM MgCl2.6H2O: 10mL/L<br />
*10% glycerol: 100 ml/L<br />
<br />
==== Hutner's Mineral Base Concentrate ====<br />
It is not difficult, but follow directions carefully.<br />
<br />
* 20g nitrilotriacetic acid (NTA)<br />
* 54.5g magnesium sulfate heptahydrate<br />
* 6.67g calcium chloride dihydrate<br />
* 18.5mg ammonium molybdate tetrahydrate<br />
* 198mg iron sulfate monohydrate (or 323.7mg of the heptahydrate)<br />
* 100mL Metals-44<br />
<br />
TO BE CONTINUED<br />
<br />
==== 5x Ligation Adjustment Buffer ====<br />
* Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer <br />
* KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0)<br />
* CaCl2 400 mM (200 ml/l of a 2 M solution) <br />
* MnCl2 100 mM (100 ml/l of a 1 M solution) <br />
* Glycerol 46.8% (468 ml/liter) <br />
* pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter) <br />
** Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --Meaganl 15:50, 25 January 2007 (EST) <br />
* water to 1 liter <br />
* autoclave or sterile filter <br />
* Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5 <br />
* Reshma 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional.<br />
<br />
=== DNA and protein gels ===<br />
==== Agar ====<br />
===== 0.8% =====<br />
# Weigh out .8g agarose<br />
# Put agarose in 500mL Erlenmeyer flask.<br />
# Add 100mL 1X TBE Buffer. Swirl.<br />
# Microwave at 100% power until the agarose starts to dissolve. Every 30-40 seconds, stop and swirl. Continue microwaving until solution boils and agarose is dissolved.<br />
# After agarose is dissolved, cover flask with foil or aran wrap and place in 55-60C waterbath until needed. <br />
# When Agarose has cooled to enough to touch comfortably, add 10microliters Syber Safe. Swirl.<br />
# Pour 30mL gels. Use 50Ml conical tube to measure 30mL.<br />
===== 1.2% =====<br />
Same as above but use 1.2 g agarose instead of .8g<br />
<br />
<br />
====Coomassie Blue Stain/Destain Recipe====<br />
<br />
450 ml water<br><br />
450 ml methanol<br><br />
100 ml 100% (glacial) acetic acid<br><br><br />
<br />
#Add 2 g per liter coomassie blue for stain. Destain is exactly the same, except do not add coomassie blue.<br />
#To stain the gel, incubate it with gentle shaking for at least 30 minutes. Pour the stain back into the bottle (you can reuse it). Rinse out the residual stain with a small amount of destain solution, and then incubate with gentle shaking for ~1 hour or more. To speed up destaining, you can put some folded KimWipes in the dish. The dye will adsorb to these, which will remove it from solution.<br />
#After your gel is destained, you can rehydrate it in distilled water and then image it and/or place it on a piece of filter paper and dry it with a gel dryer.<br />
<br />
== Protocols ==<br />
=== Growing Cells ===<br />
==== TOP10 Competent Cells ====<br />
* Prechill plasticware and glassware<br />
<br />
===== Preparing seed stocks =====<br />
# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C <br />
#* room temperature works well <br />
# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C <br />
#* room temperature works well <br />
# Add glycerol to 15% <br />
# Aliquot 1 ml samples to Nunc cryotubes <br />
# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes <br />
#* This step may not be necessary <br />
# Place in -80°C freezer indefinitely.<br />
<br />
===== Preparing competent cells =====<br />
# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 <br />
#* This takes approximately 16 hours. <br />
#* Controlling the temperature makes this a more reproducible process, but is not essential. <br />
#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule <br />
#* Aim for lower, not higher OD if you can't hit this mark <br />
# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle. <br />
#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend <br />
#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer <br />
# Gently resuspend in 80 ml of ice cold CCMB80 buffer <br />
#* sometimes this is less than completely gentle. It still works. <br />
# Incubate on ice 20 minutes <br />
# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer. <br />
# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells. <br />
# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. <br />
# Incubate on ice for 20 minutes <br />
# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates <br />
# Store at -80°C indefinitely. <br />
#* Flash freezing does not appear to be necessary <br />
# Test competence (see below) <br />
# Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. <br />
<br />
===== Measurement of competence =====<br />
# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) <br />
#* This is at 10 pg/_l or 10-5 _g/_l <br />
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE <br />
# Hold on ice 0.5 hours <br />
# Heat shock 60 sec at 42C <br />
# Add 250 _l SOC <br />
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated <br />
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. <br />
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tetracycline resistant, we find growing for 2 hours yields many more colonies <br />
#* Ampicillin and kanamycin appear to do fine with 1 hour growth <br />
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads <br />
#* Good cells should yield around 100 - 400 colonies <br />
#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA <br />
#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA <br />
<br />
===== Glycerol Stocks =====<br />
<br />
====== Materials ======<br />
* 40% glycerol solution <br />
* Cryogenic vials <br />
<br />
====== Method ======<br />
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial. <br />
# Add 1 ml sample from the culture of bacteria to be stored. <br />
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. <br />
#* Alternatively, pipet to mix. <br />
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. <br />
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. <br />
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. <br />
<br />
====== Notes ======<br />
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.<br />
Prof. Schonbaum's version<br />
# Grow a 3 ml culture overnight<br />
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols<br />
# Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.<br />
<br />
=== DNA prep ===<br />
<br />
====Zyppy mini-prep====<br />
<br />
The following procedure is performed at room temperature. <br />
Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash <br />
Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, <br />
incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion). <br />
<br />
#Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube<br />
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. '''1B)''' Discard the supernatant. '''1C)''' Repeat steps 1A and 1B as needed. '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely. <br />
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes. <br />
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds. <br />
# Discard the flow-through and place the column back into the same Collection Tube. <br />
# Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube. <br />
# Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds. <br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature. <br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
'''Notes'''<br />
<br />
# Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been <br />
neutralized and mixed thoroughly. <br />
# Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer. <br />
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�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral <br />
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