Making your own electrocompetent cells
Innoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask .
Incubate for 16 - 18 hours at 37°C and 250 rpm .
Pre - warm 2 , 1 L flasks containing 250 ml each of SOB .
Add two drops of the overnight culture to each of the pre - warmed 250 ml flasks .
Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5 - 0.7 .
Turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells .
Place 1 L of 10 % glycerol on ice well in advance of harvesting cells .
Place cultures on ice for 15 minutes .
From this point on the cultures must be kept ice cold .
Pour each 250 ml culture into chilled 500 ml ( or 1000 ml ) centrifuge bottles .
Centrifuge at 5000 rpm for 10 min .
Pour off the supernatant and aspirate any residual broth .
Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down .
Centrifuge at 5000 rpm for 10 min .
Pour off the supernatant , it is not necessary to aspirate .
Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down .
Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down .
To freeze , add 100 µl of the culture to microcentrifuge tubes on ice .
Once you have used all of the culture , transfer the tubes to dry ice for 10 minutes .
Once the cultures are frozen , transfer them to a - 80°C freezer
