Team:Stockholm/18 July 2010

From 2010.igem.org


Contents

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

  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.





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligof├Ârbundet) Geneious Fermentas/ Sigma-Aldrich/