Paris/September 21
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| + | [[Paris/September 20|yesterday]] -- [[Paris/September 22|tomorrow]] <br> | ||
==Electro-competent cells for wanner deletion of DapA== | ==Electro-competent cells for wanner deletion of DapA== | ||
* 100ml culture of MG1655 pKD46 from ON cult. | * 100ml culture of MG1655 pKD46 from ON cult. | ||
Latest revision as of 18:06, 7 October 2007
Electro-competent cells for wanner deletion of DapA
- 100ml culture of MG1655 pKD46 from ON cult.
- grow until DO=0.2
- add arabinose to a final concentration of 0.4%
- grow until DO=0.6
- keep on ice for 10min
- centrifuge 10min at 4000rpm and 4°C
- resuspend in 50ml cold H2O
- centrifuge 10min at 4000rpm and 4°C
- resuspend in 50ml cold H2O
- centrifuge 10min at 4000rpm and 4°C
- resuspend in 25ml cold 10% glycerol
- centrifuge 10min at 4000rpm and 4°C
- resuspend in 200µl cold 10% glycerol
- keep on ice until transformation
Transformation
- Transformation of the electrocompetent cells with the PCR product made on pKD13
- Selection on Km + DAP
