Competent cells

Take a single colony from a fresh plate and grow a 10 ml pre-culture overnight at 37oC. Don't forget that XL1 / XL10 cells have an intrinsic antibiotic resistance (chloramphenicol / tetracyclin). Cells growing without antibiotic (eg BL21) double approx every 20 mins, those with antibiotic double approx every 30mins.

  • Inoculate 1l culture with 5-10ml preculture and grow at 37oC until OD600=0.3.

  • Cool on ice for 30 mins

  • Centrifuge in autoclaved centrifuge tubes at 5000rpm for 10mins (remember, not all plastic centrifuge pots can be autoclaved - our 500ml ones can, the communal 1l ones cannot)

  • Remove supernatant and resuspend pellet in ice cold 0.1M CaCl2 / 25% glycerol by volume (0.5l per 1l of medium). Resuspend gently by pipetting up and down - do not vortex!

  • Centrifuge and resuspend again

  • Centrifuge and resuspend in 2-10ml CaCl2 / glycerol

  • Dispense into eppendorf tubes (30-50ul / tube)

  • Flash freeze in liquid nitrogen and store at -70oC