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