Paris/September 21

From 2007.igem.org

yesterday -- tomorrow

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