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

  1. Damon needs the shakers at 30C, so will have to put off redo of competent cells. Will help him

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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