Making Chemically Competent E. coli Cells

Reagents and Materials

• Top10 E. coli cells
• LB Broth
• 50mM CaCl₂ solution (store at 4 degrees Celsius)

Protocol

1. Inoculate 5-10 mL LB at 37 degrees Celsius while shaking
2. Subculture 1 mL of bacteria solution into 50 mL LB broth at 37 degrees Celsius; 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 degrees Celsius for 2 minutes
4. Re-suspend 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 degrees Celsius for 2 minutes and re-suspend 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 degrees Celsius