Protocols

Creating Competent Cells

Materials
LB Media, 50ml Falcon Tubes, Ice, Chilled centrifuge, Calcium Chloride (CaCl2), Eppendorf tubes (300ul/tube)

Procedure
Inoculate a single colony into 5ml Lb in 50ml falcon tube. Grown O/N @ 37oC
Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.
Shake @ 37oC for 1.5-3 hours.

Or

Inoculate a single colony into 25ml LB in a 250ml bottle in the morning
Shake @ 37oC for 4-6 hours.

Then…

Put the cells on ice for 10mins (keep cold from now on).
Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm.
Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption).
Incubate on ice x 20 minutes
Centrifuge as in 2.
Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol.
Dispense in microtubes (300ųl/tube). Freeze at -80oC.