Team:University of Chicago/Notebook/Norayucel

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Revision as of 17:39, 29 July 2008

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:.006
- 11:52: .017
- 1:50: .033

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)

@@ Line 34: / Line 34: @@
 ==July 22, 2008==
-'''We have cells!!'''
+*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''':.006
+#*'''11:52''': .017
+#*'''1:50''': .033<br><br>
+'''TOP 10'''
 #10pg: 3 colonies.
 #100pg: 25 colonies
@@ Line 44: / Line 50: @@
 #*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==