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)
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