Making Chemically Competent E. coli Cells

This procedure was done using Top10 cells ordered from Invitrogen. 50 mL Falcon tubes were used for this protocol.

1. Innoculate 5-10 mL LB at 37°C while shaking
2. Subculture 1 mL of bacteria solution into 50 mL LB broth at 37° while shaking until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours)
3. Centrifuge the subculture at 10 000 rpm at 4°C for 2 minutes
4. Resuspend pellet in 12.5 mL of cold CaCl₂ (50 mM) and leave on ice for 10 minutes
5. Centrifuge at 10 000 rpm at 4°C for 2 minutes and resuspend in 2 mL of cold CaCl₂ (50 mM, 15% glycerol solution)
6. Leave on ice for at least 30 minutes and then aliquot 200 μL and freeze at -80°C