Paris/July 26
From 2007.igem.org
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== Microscopic DAP complementation experiment == | == Microscopic DAP complementation experiment == | ||
| - | We used wild type E.coli transformed by part J23107 (RFP) and '' | + | 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. |
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| + | 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. | ||
Revision as of 11:05, 6 August 2007
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.
