Team:Stockholm/18 July 2010

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(Difference between revisions)

Revision as of 20:56, 29 July 2010

Andreas

BL21 restreaks

IPTG-induced BL21

Results from 17/7 restreaks

Nice fluorescent cell colonies appeared on the plate, indicating that IPTG induction from the pEX vector indeed works and that I have successfully inserted the three fluorescent protein genes into pEX.

Competent Top10

Continued from 17/7 At 9:30 AM cell culture still hadn't reached OD₆₀₀ 0.3, only 0.02. I therefore transfered the cells to 37°C, 250 rpm until an OD₆₀₀ of 0.3. I then followed our standard Top10 protocol.

Colony PCR of pEX constructs

Continued from 17/7

A new colony PCR was run on the same clones as were picked 17/7, using the same tube settings except (1) 1.5 μl primer was used instead of 1.0 μl and (2) 1 μl cell suspension was used instead of 0.5 μl; dH₂O volume was decreased accordingly. Samples pEX.SOD A and pEX.SOD B were omitted.

PCR settings

95°C - 5:00
95°C - 1:00
60°C - 0:30
72°C - 1:15 (Cycle to step 2. 29 times)
72°C - 5:00
15°C - ∞

Gel verification

1 % agarose, 90 V, 40 min

Results

Not all bands present in all lanes. Due to lack of time, all samples were discarded and I will pick/verify new colonies after my vacation.

@@ Line 16: / Line 16: @@
 ''Continued from 17/7''
-A new colony PCR was run on the same clones as were picked 17/7, using the same tube settings except (1) 1.5 &mu;l primer was used instead of 1.0 &mu;l and (2) 1 &mu;l cell suspension was used instead of 0.5 &mu;l; dH<sub>2</sub>O volume was decreased accordingly. Samples pEX.SOD A and pEX.SOD B were ommitted.
+A new colony PCR was run on the same clones as were picked 17/7, using the same tube settings except (1) 1.5 &mu;l primer was used instead of 1.0 &mu;l and (2) 1 &mu;l cell suspension was used instead of 0.5 &mu;l; dH<sub>2</sub>O volume was decreased accordingly. Samples pEX.SOD A and pEX.SOD B were omitted.
 '''PCR settings'''