Electrocompetent Cells

• inoculate 1L SOB-Medium with 1ml E.coli overnight culture and place 2l 10% glycerol on ice
• wait for OD 0,6-0,8
• put culture ice for a temperature of 0-4°C
• distribute in 6 centrifugation-bottles (250 ml)
• spin 10 min at 2500-4000 rpm
• resuspend the Pellets with 1l ice-cold 10% glycerol
• spin 10 min. at 8000 rpm
• resuspend Pellets with 1l ice-cold 10% glycerol, keep some 10% glycerol
• spin 10 min at 8000 rpm
• resuspend Pellets with 50ml 10% glycerol
• spin 10 min. at 8000 rpm
• resuspend Pellets in low quantity of 10% glycerol
• aliquot into eppendorf-tubes, freeze in liquid N₂ and keep at -70°C

Now the cells are able to pick up plasmids. With electroshocks the membrane becomes porous.