Preparation of Chemically Competent Cells

Equipment

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

Inoculate 50 mL of RB in Klett flask with 0.5 mL of culture.
Grow to an optical density of 0.5 - 0.7 @ 600 nm wavelength.
Centrifuge 8000 rpm 5 min in sterile JA-17 tubes.
Resuspend in 5 ml ice-cold CaCl2; you can vortex at this step. Probably stay gentile…
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. Be very gentile at this point. NEB recommends that you flick the tube gently with your finger to do this.
Aliquot 50 uL into single-use eppendorf tubes. Should get 30 total.
Flash freeze with liquid nitrogen.

Test competency by transforming pUC19 and then doing the serial dilution plating.

Streak/plate bacteria of choice on LB agar plate.
Inoculate single colony into starter culture of 20 mL SOC media in 125 mL Erlenmeyer flask.
Incubate overnight in 30C or 37C shaker.
Inoculate growth culture 1:100 with starter culture. (2.5 mL of starter into 250 mL 2XYT media in 1L Erlenmeyer flask). Run in 37C shaker.
Grow until OD600 reaches 0.4 to 0.6 (~5h).

After this point, keep everything cold. Work in cold room and pre-chill all supplies.

Transfer bacteria to culture flasks and spin down. 5000xg, 4C, 10 min.
Pour out supernatant.
Gently rinse flasks and pellet with small aliquot of TFB1 to remove all traces of media.
Add 100 mL TFB1 per 250 mL of growth culture and resuspend using 10 mL serological pipette.
Incubate in wet ice 5 minutes.
Spin down 5000xg 4C 5 min.
Remove all supernatant.
Add 10 mL TFB2 per 250 mL growth culture and gently resuspend by serological pipette.
Incubate in wet ice 15-6- mins.

Dispense 100 uL into pre-chilled 1.5 mL micro centrifuge tubes and snap freeze in LN2.
Store at -80 C.