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Team:University of Chicago/Notebook/Norayucel
From 2008.igem.org
July 18, 2008
1. Diluted 1ng/microliter pGreen plasmid to 10pg/microliter with 100X dilution
2. 1microliter of plasmid to 50 microliters competent cells
3. Will grow up 50microliters of TOP10 untransformed as a control
4. Put on ice at 2:03
5. Took off ice at 2:34
6. Incubated for 1minute at 42C
7. Added 250microliters SOC media (prepared at 1:30)
8. Start incubated at 37C at 2:36
9. Take out at 3:40pm
10. Streaked 20microliters onto Amp. Plates
- +pGreen
- NO plasmid
11. Dan will come in Saturday morning to pick up the plates and count colonies.
July 21, 2008
- No colonies grew on 10pg/microliter.
- Try transformation again before trying to grow up new TOP10
- DNA could be bad
- Wrong antibiotic (double checked: ampicillin is correct)
- Something went wrong with transformation--perhaps too long at 42C?
- Bad cells (nooooo....)
Retry
- 10pg/microliter
- 100pg/microliter
- 1ng/microliter
- no plasmid on LB (test if cells are completely dead)
Also try Dr. Schonbaum's stocks to compare
- 10pg/microliter
- 1ng/microliter
transformed at 4pm and put in 37C incubator. Check tomorrow morning.
July 22, 2008
- Checked OD of Damon's caulobacter. OD should be around 1.1. Is actually at .006.
- Will track OD during day until Damon comes in
- 9:45 OD:0.006
- 11:52: 0.017
- 1:50pm: 0.033
- Damon and I will come in later to check on OD and dilute to appropriate OD so OD tomorrow morning is ~1.1.
TOP 10
- 10pg: 3 colonies.
- 100pg: 25 colonies
- 1ng: 200 colonies
- Efficiency of cells is ~3x10^6. Need to redo.
- NO plasmid on LB: big streaky mess. Cell mos' def' alive.
- STOCKS, 10pg: 22
- Stocks, 1ng: 300-400
- Weird. Should be 100X more than 10pg. Perhaps bad dilution.
- Efficiency going off 10pg is 3-4x10^7. Sadly Dr. Schonbaum's stocks aren't efficient enough. What to do....
July 23, 2008
- Damon needs the shakers at 30C, so will have to put off redo of TOP10.
Check OD
- 10:00am: OD is 1.43.
- 10:45: OD is .14.
- Dilute to .14
- ~700mL is 2L flask
- 1:45pm: OD is .39
July 24, 2008
- Set 3mL starter culture at ~6pm.
July 25, 2008
- Remade TOP10 competent cells--will try to make more competent
- Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture
- Two 2L flasks with 250mL each
- At 5:30 OD was .28 and .29 for each flask respectively
- Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)
- Put into -80C at 7:30pm.
July 28, 2008
Transformation with pGreen
- 10pg/microliter on LB Amp
- 1 ng/microliter on LB Amp
- NO plasmid on LB Amp
- NO plasmid on plain LB
- Put into 37C incubator ~1:30pm
Plates
- Running out of plates
- 500mL of LB and LB Amp agar
- 100mg/mL stock of Ampicillan
- After cooling for ~45 minutes at room temp, added .5mL
- Poured around 4:30 pm. Turned over at 5:30
Checked plates before I left at ~6pm. No growth (as expected). Will look again in the morning
July 29, 2008
- Checked plates
- 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(
- 1ng/microliter plate was a big streaky mess (too many to count)
- NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??
- NO plasmid +LB big streaky mess (as expected)
August 5, 2008
Transformations with DH5alpha
Old LB/Amp plates -plasmid: 200microliters +pGreen: 10microliters +pGreen: 50 microliters +pGreen: 250 microliters
New LB/Amp plates -plasmid: 200 microliters +pGreen: 50 microliters
LB -plasmid +pGreen
August 6, 2008
RESULTS
DH5-ALPHA (plated on 8.5.08) Old LB/Amp plates -strange film on all the old LB/Amp plates. Contamination? -plasmid: Only haze
- +pGreen 10microliters: haze, with a few blips that are not green
- +pGreen 50 microliters: haze, more blips, but still not green
- +pGreen 250microliters: haze+non-green blips
New LB/Amp plates
- -plasmid: Nothing.
- +pGreen 50 microliters: nothing
LB
- -plasmid: nothing
- +pGreen 50 microliters: lots of cells
Transformations iGEM:
- TOP10+p1010/LB: smear
- TOP10+E02040/Amp: nothing
- TOP10+pGreen (125microliters): ~20 colonies
- TOP10+TE buffer: smear
August 7,2008
RESULTS from iGEM (8.6.08)
- TOP10+p1010/LB: Lots of cells
- TOP10+E02040/Amp: nothing
- TOP10+pGreen (125microliters): 12 very small green colonies
- TOP10+TE buffer:Lots of cells
..Results don't look so hot. Lets try the transformation again.
TEST: TOP10+colony of pGreen transformed from 7.28. Maybe Amp plates are bad?
Plates
- TOP10+E2040/Amp
- TOP10+CFP/Amp
- TOP10/LB:
- TOP10+pGreen
August 8, 2008
RESULTS from 8.7.08 transformation
- TOP10+E2040/Amp: Nothing
- TOP10+CFP/Amp:Nothing
- TOP10/LB: Lots of cells (so cells are viable)
- TOP10+pGreen: 40 colonies
August 11, 2008
- Retry pGreen/DH5alpha with Schonbaum's Amp plates
- Made new amp plates with 250mL agar LB +250microliters of 100mg/mL ampicillin (fresh)
August 13/14 at PCBio retreat. Whoo!
August 18
- Grew up 250mL of DH5-alpha in LB
- Prepped using Thomas’s protocol
- Grew for three hours, stopped at OD of .301
- Total volume ~800microliters
August 19, 2008
Grew up 250mL of XL21 in SOB
- had trouble before getting starter culture to work, so made 4 starter cultures, one of which had multiple colonies. *Only three grew
- Start shaking at 9:30, took out at 1:20. (OD of .35)
- Electroporation of GFP generator with DH5alpha
- pGreen (1microliter of 10pg/microliter)
- E0240
- Using stock cells+E0240
- Had to do pGreen twice, first time time constant of 8.76. Second time was 4.82
- With E0240 time constant was 8.22 (?)
- Stock cells popped—junked it.
- Added 1mL of SOC plated 80microliters for pGreen, 1mL for E0240 (scaled up to accommodate for extra media dilution)
August 20,2008
- DH5alpha at competency of ~5x10^7 (15 colonies)
- Still no iGEM plasmids. What gives?
- Used fluorometer to check concentration of iGEM part from well 12A and control filter paper plasmid
**iGEM at 80 picograms/microgram, control is at 72
- Add one microliter of solution of 10microliters to 50 microliters DNA
- Electroporate
- All time constants between 4.6 and 4.9.
August 21, 2008
- XL1 BLUE ARE COMPENTENT! ~320 colonies
- iGEM still doesn’t transform. What gives?!
- Check caulobacter: it’s caulobacter!!! Looks all pretty and curvy.
- Set up culture for miniprep of mystery plasmid (100mL LB+.1mL chloramphenicol stock of 35mg/mL)
- streaked one colony of mystery plasmid on chloramphenicol plate
August 22, 2008
- Overnight culture didn’t grow, however plate has a few colonies
- Will keep growing mystery plasmid culture. 2 3mL of plate colonies, and another 1 3mL tube of old plate colonies.
- MINIPRPEP!
- Stabs arrived from E0240 and OompX
- Streak
cacc tagaatatca
61 ttttcgccct ctgatattct atttggtgtt ctagatcgct tgttcaaaga taacgctacc
121 gggaaggttc ttgcttcccg ggtagctgtc gtaattcttt tgtttataat ggcgattgtt
181 tggtataggg gagatagttt ctttgagtac tataagcaat caaagtatga aacatacagt
241 gaaattattg aaaaggaaag aactgcacgc tttgaatctg tcgccctgga acaactccag
301 atagttcata tatcatctga ggcagacttt agtgcggtgt attctttccg ccctaaaaac
361 ttaaactatt ttgttgatat tatagcatac gaaggaaaat taccttcaac aataagtgaa
421 aaatcacttg gaggatatcc tgttgataaa actatggatg aatatacagt tcatttaaat
481 ggacgtcatt attattccaa ctcaaaattt gcttttttac caactaaaaa gcctactccc
541 gaaataaact acatgtacag ttgtccatat tttaatttgg ataatatcta tgctggaacg
601 ataaccatgt actggtatag aaatgatcat ataagtaatg accgccttga atcaatatgt
661 gctcaggcgg ccagaatatt aggaagggct aaataatta
Order: LL-H Lysin/thymol Princeton apoptosis gene? Maybe think about that later!
August 23, 2008
-Both stocks of pUC8 have cells. Whoo! -How to deal with bacterial stab?
August 24, 2008
-Put stabs in 37C incubator.
August 25, 2008
- Miniprep DNA, digest with BamHI and EcoRI
- Run gel
-Lane 1: 1kb ladder -Lane 2: Plasmid stock 1 -Lane 3: Plasmid stock 2 -Lane 4: DWP1 (miniprepped plasmid)
- band. Plasmid definitely present.
- 3 mL culture of bacterial stabs, plates. Put in incubator at 5pm.
- Create biobricks for mefp-5 caulobacter and E. coli
August 26, 2008
- both iGEM stocks grew up
- GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)
- Ran on 1.2% agarose gel. 5microL DNA/1microL dye.
- Very strong bands
- Restriction digest of pUC8/caulobacter secretion plasmid. Incubate at 37C for 1hour.
- Use EcoRI, HindIII, SolI, BamHI
Buffers
-Enzyme EcoRI HindIII SolI BamHI -Buffer (10x) H B H B
Materials/amounts
-DNA (L) Buffer (B or H, microL) MilliQ water (microL) Enzyme (microL) -Single Digest 2.5 2 15 0.5 -EcoRI/HindIII 2.5 2 (B) 14.5 0.5+0.5 -EcoRI/SolI 10 2 (H) 7 0.5+0.5
Agarose Gels (for each stock, 1 or 2)
- 5 microL rxn mixture/1microL loading dye
- 15microL ladder
-Lane 1 2 3 4 5 6 7 8 -Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI
August 27, 2008
- Two bands for SolI, strange cuts in EcoRI/Sol.
- Stocks 1 and 2 are the same.
- Redo. Try old SolI stock and a new aliquot.
- Send to sequencing with M13 primers
- DNA (microL) Buffer (H, microL) MilliQ water (microL) Enzyme (microL) -Single Digest 1.5 2 16 0.5 -EcoRI/SolI 8 2 (H) 9 0.5+0.5
-Lane 1 2 3 4 5 6 7 -Contents 1kb ladder NO enzyme SolI (old) SolI (new) EcoRI/SolI (old) EcoRI/SolI (new) pBluescript (check)
- do Restriction digest with GFP and OmpX?
August 28, 2008
PC-Bio presentation!
- 442micrograms/microliter concentration (Stock 2 of pUC8 mystery plasmid)
- No explanation as to why SolI is cutting twice. Submit to sequencing facility using M13Rev primer
August 29, 2008
Made BL21 competent cells with Rob (for pLysS). ~60 50microliters vials.
August 30, 2008
- 50microliters cells/1 microliter pGreen
- Add 100microliters SOC after electroporating, shake at 37C for one hour
- Plate 80microliters on plates
- Test transformation efficiency
- On LB+Amp plates
- BL21+10pg/microliter pGreen: 20microliters
- BL21+100pg/microliter pGreen: 20 microliters
- BL21 --: 20 microliters
- On LB plates
- BL21 --: 20 microliters
August 31, 2008
- Number of colonies
- 100pg: 100 colonies,
***Efficiency is 1.3x10^7 transformants/microgram of DNA
- 10pg had only ~3 colonies (?)
- No plasmid: Grew up on LB, didn’t on amp (so that checks out)
Sept 2, 2008
- Checked extra pUC8. Prep was fine.
- Sequencing results show plasmid is pUC8 CVX. Whoo!
Sept 3, 2008
iGEM inventory (sent 8/28)
- I20260 K –Measure ment kit test of J23101. (promoter +GFP, standard promoter)
- I20269 K—J23150 (weak) promoter+GFP
- I20270 K--
- P1010-pSB3K3
- J23101 A
- P1010—pSB1A2
Sept 5, ,2008
- GROWING CAULOBACTER!
- 5ml O/N culture of pUC8 CVX transformed bacteria from damon’s plate’s (july 23).
- 100X dilution, with loop
- 100X dilution, with pipette tip
- 10X dilution, with loop
- 10X dilution, with pipette tip.
Tried own transformation with sequenced S2, pUC8 CVX DNA.
- 3microliters of DNA in 50microliters competent cells.
- Time constant was 4.84, 4.90 for + and – plasmid respectively
- Added 100microliters PYE following transformation, shook at 30C for 2.5 hours
-Plate 1: PYE/Chlr. Caulobacter+pUC8, 100microliters -Plate 2: PYE/Chlr. Caulobacter+pUC8, 10microliters -Plate 3: PYE/Chlr. NO plasmid -Plate 4: PYE NO plasmid
- Come back tomorrow morning ~8 to pick up culture/plates.
- Rob is making media necessary for growth. Yay Rob!
