Team:University of Chicago/Notebook/Norayucel

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Contents

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

  1. No colonies grew on 10pg/microliter.
  2. 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?
  3. 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.
  1. Will track OD during day until Damon comes in
    • 9:45 OD:0.006
    • 11:52: 0.017
    • 1:50pm: 0.033
  2. 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

  1. 10pg: 3 colonies.
  2. 100pg: 25 colonies
  3. 1ng: 200 colonies
  • Efficiency of cells is ~3x10^6. Need to redo.
  1. NO plasmid on LB: big streaky mess. Cell mos' def' alive.
  2. STOCKS, 10pg: 22
  3. 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

  1. 10:00am: OD is 1.43.
  2. 10:45: OD is .14.
    • Dilute to .14
    • ~700mL is 2L flask
  3. 1:45pm: OD is .39

July 24, 2008

  1. Set 3mL starter culture at ~6pm.

July 25, 2008

  1. Remade TOP10 competent cells--will try to make more competent
  2. Inoculated 500mL of SOB at ~10am with 1mL each of TOP10 starter culture
    • Two 2L flasks with 250mL each
  3. At 5:30 OD was .28 and .29 for each flask respectively
  4. Final OD after following competent cell protocol was 1.04 (between 1-1.5 as required)
  5. Put into -80C at 7:30pm.

July 28, 2008

Transformation with pGreen

  1. 10pg/microliter on LB Amp
  2. 1 ng/microliter on LB Amp
  3. NO plasmid on LB Amp
  4. NO plasmid on plain LB
  5. Put into 37C incubator ~1:30pm

Plates

  1. Running out of plates
  2. 500mL of LB and LB Amp agar
    • 100mg/mL stock of Ampicillan
    • After cooling for ~45 minutes at room temp, added .5mL
  3. 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

  1. Checked plates
  2. 10 pg/microliter plate had ~20 colonies. Competency still off by factor of 10 :(
  3. 1ng/microliter plate was a big streaky mess (too many to count)
  4. NO plasmid on LB+Amp also big streaky mess. Possibly mislabeled plate??
  5. 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

  • 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

  • both iGEM stocks grew up
  • GFP generator is ~3kb, OompX is ~4kb (note when miniprepping)

**Ran on 1.2% agarose gel. 5L DNA/1L 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, L) MilliQ water (L) Enzyme (L) 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 L rxn mixture/1L loading dye -15L ladder Lane 1 2 3 4 5 6 7 8 Contents 1kb ladder NO enzyme EcoRI HindIII SolI BamHI EcoRI/HindIII EcoRI/SolI

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