From 2007.igem.org

Colonies PCR for cloning verification

L1.1, L1.2, L2.1, L2.2, L9.1, L10.1

L6: 8 clones

Toothpick in 50µl sterile water ==> 10'@100°C

PCR Mix:

• 212.5 µl Master Mix
• 8.5 µl VF2
• 8.5 µl VR

12 µl of the mix in 13µl of lysed cells for each PCR reaction

Cycle x30:

• 95°C 30
• 60°C 30
• 68°C 2'

Microscopic DAP complementation experiment

We used wild type E.coli transformed by part J23107 (RFP) and dapA- E.coli (w121) auxotroph to DAP. E.coli transformed by part J23107 (RFP) is able to synthesize DAP and export it. We suppose dap- E.coli could be feed by wt E.coli.

We tried to observe kinetics by fluorescence microscopy. We let separately in culture overnight dapA- E.coli (LB/erythromycine/DAP) and rfp wt E.coli (LB ampicillin). The day after, we diluted both liquid culture 1/200 to have exponential culture. Then we put on slide different conditions:
- 50% wt rfp / 50% dapA- (colomn 1)
- 70% wt rfp / 30% dapA- (colomn 2)
- 30% wt rfp / 70% dapA- (colomn 3)
- 90% wt rfp / 10% dapA- (colomn 4)
- 10% wt rfp / 90% dapA- (colomn 5)