Team:Stockholm/17 July 2010

From 2010.igem.org

Andreas

LMWP ligations

Continued from 16/7

No colonies on any of the plates - unclear why. New ligation strategy will be tried, but not right now as we're out of ligase.

Glycerol stocks of verified pSB1C3 constructs (16/7)

3 ml LB + 25 μg/ml Cm inoculated with previously picked colonies for colony PCR (16/7). Cultures grown in 37°C, 250 rpm. Unfortunately the marked text disappeared from tubes, so new ON cultures had to be set. These were grown in 30°C, no rotary shaking.

Colony PCR of pEX constructs

Since previous colony PCRs of Top10/pEX were unsuccessful, a new colony PCR was attempted. Colonies were picked from 13/7 plates and dissolved in 10 μl LB:

pEX.BBa_J18930 A
pEX.BBa_J18930 B
pEX.BBa_J18931 A
pEX.BBa_J18931 B
pEX.BBa_J18932 A
pEX.BBa_J18932 B
pEX.SOD A
pEX.SOD B

PCR tubes - illustra PuReTaq Ready-To-Go™ PCR Beads:

1 μl 10 μM forward primer (pEXf)
1 μl 10 μM reverse primer (pEXr)
0.5 μl LB cell suspension
22.5 μl dH₂O

PCR settings

95°C - 5:00
95°C - 0:30
60°C - 0:30
72°C - 1:30 (Cycle to step 2. 29 times)
72°C - 10:00
15°C - ∞

Results

No good gel results. Gel and samples discarded.

Plasmid prep. from Top10 pSB1C3.X ON cultures

Continued from 16/7

Plasmid prep. procedures as previously described.

Elution volume: 70 μl. Stored in -20°C for later DNA conc. measurements.

BL21 restreaks

It is quite strange that I couldn't get my cultures to fluoresce (15/7). On Sridhar's suggestion, I picked one new colony from each of the three BL21 plates with pEX.BBa_J18930-32 constructs and restreaked on a 100 μg/ml Amp plate treated with 50 μl 0.1 M IPTG, each construct on 1/3 of the plate. Plate grown ON in 37°C.