Making your own chemically competent cells


Fresh overnight culture of desired strain grown in RB (Rich broth = Luria-Bertani broth)
40 ml sterile centrifuge tubes (e.g. Beckman JA-17 rotor)
Sidearm flask (or other 250mL shaker flask)
Klett meter (or OD600 spectrophotometer)
Ice-cold 30 mM CaCl2
37°C water bath
18x150 mm capped culture tubes
Eppendorf tubes
Rollerdrum or shaker
Selective plates


Subculture bacterial overnight 0.5 ml/50 ml RB in Klett flask. Grow to 60-75 Klett units (0.5 – 0.7 A600). Centrifuge 8000 rpm 5 min in sterile JA-17 tubes. Resuspend in 5 ml ice-cold CaCl2; you can vortex at this step. If convenient, the cells can be left on ice at this point for several hours.

When ready to use, distribute 1.5 ml to three Eppendorf tubes and spin 30 seconds in a microcentrifuge. Resuspend each pellet in 0.5 ml ice-cold CaCl2, without vortexing. Flick the tube gently with your finger to do this.

Aliquot 50 uL into single-use eppendorf tubes.

Add DNA, usually 1 uL of miniprep DNA, 2 of ligation mixture, or an appropriate dilution of cesium-purified DNA. Incubate on ice 30 min. Heat shock at 42°C for 30sec, then chill again on ice 5 min. Outgrow in 1 ml RB for 1 hour at 37°C and plate on selective plates.

Rich Broth

Per liter
10 grams Tryptone
5 grams Yeast Extract
5 grams NaCl
2 ml NaOH pH 7.2