Making electro-competent cells

Streak the cells on LB agar plates and incubate at 37°C for 12h 0m 0s

Pick single colony and inoculate it in 20mL 2X LB broth and incubate at37°C for 12h 0m 0s

• 16h 0m 0s on shaker incubator

Take 2ml of primary inoculum and inoculate it in 200mL 2X LB broth and incubate it at 37°C

on shaker.

Grow till the OD reaches 0.6

Place the culture at 4°C for 1h 30m 0s

Aliquot in50mLfalcon and spin at 4000rpm,4°C

Discard the supernatant and resuspend well in 40mL 10% glycerol

Spin at 4000rpm,4°C

Discard the supernatant and resuspend in 25mL 10% glycerol

Spin at 4000rpm,4°C

Discard the supernatant and resuspend in20mL 10% glycerol. Mix the contents of 2 falcons together such that only 2 falcons of 40mL contents is present

Spin at 4000rpm,4°C

Discard the supernatant and resuspend in1mL 10% glycerol.

Make aliquots of 50µL in autoclaved 600µL eppendorfs,flash freeze and store at -80°C