Protocol for Making Chemically (CaCl2) Competent E. coli
Inoculate 3 ml LB + appropriate antibiotic with a single colony of E. coli strain
harboring the plasmid to be isolated. Grow at 37oC for overnight with vigorous
shaking.
In the morning inoculate 25 ml LB + appropriate antibiotic with 200 l of the
overnight culture. Grow at 37oC overnight with vigorous shaking until the
culture reaches an OD A600 of 0.3-0.5 (depending on the strain this takes 2-4
hours).
Place culture on ice for 10 minutes.
Split culture into round bottom falcon tubes 4 x 6 ml.
Centrifuge tubes for 10 min at 4000 rpm a 4oC.
Decant supernatant and resuspend in 3 ml ice cold sterile 100 mM CaCl2. Let
stand on ice for 30 minutes.
Centrifuge tubes for 10 min at 4000 rpm a 4oC.
Decant supernatant and resuspend in 400 l ice cold sterile 100 mM CaCl2. Split
into eppindorf tubes 200 l cells per eppindorf tube. Cells are now competent.
The cells will remain competent for 24 hours with reducing transformation
efficiency over time. It is possible to snap freeze cells and store for longer at 80oC; however, I do not recommend this.
Transformation protocol
Take 200 l aliquot of cells from previous step and add appropriate quantity of
DNA (20 ng for supercoiled plasmids). Incubate on ice for 30 minutes.
Place tube in a 42oC waterbath for exactly 90 seconds.
Remove from waterbath and place on ice.
Add 1 ml LB media (no antibiotics) to the tube and place the tube in a 37oC
shaker (225 rpm) for an hour.
Plate an appropriate volume of the transformation reaction on LB + appropriate
antibiotic(s). Grow O/N at 37oC.
Robertson Lab