Protocol

E. coli Calcium Chloride competent cell protocol

   1. Inoculate a single colony into 5mL Lb in 50mL falcon tube. Grow O/N @ 37°C.

   2. Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning.

   3. Shake @ 37°C for 1.5-3hrs.

Or

   1. Inoculate a single colony into 25mL LB in a 250 mL bottle in the morning.

   2. Shake @ 37°C for 4-6 hrs.

Then….

   1. Put the cells on ice for 10 mins (keep cold form now on).

   2. Collect the cells by centrifugation in the big centrifugue for 3 mins @ 6000rpm

 3. Decant supernatant and gently resuspend on 10 mL cold 0.1M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).

   4. Incubate on ice x 20 mins

   5. Centrifuge as in 2

   6. Discard supernatant and gently resuspend on 5mL cold 0.1MCaCl/15%Glycerol

   7. Dispense in microtubes (300μL/tube). Freeze in -80°C.