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Team:University of Chicago/Notebook/Norayucel
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*Checked OD of Damon's caulobacter. OD '''should''' be around 1.1. Is actually at .006. | *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 | #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''' | '''TOP 10''' | ||
Revision as of 17:42, 29 July 2008
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
- 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 competent cells. Will help him
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)
