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From 2010.igem.org

Contents 1 Andreas 1.1 BL21 restreaks 1.2 Competent Top10 1.3 Colony PCR of pEX constructs 1.3.1 Results

Andreas

BL21 restreaks

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

1. 95°C - 5:00
2. 95°C - 1:00
3. 60°C - 0:30
4. 72°C - 1:15 (Cycle to step 2. 29 times)
5. 72°C - 5:00
6. 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.