Yu:Competent Cells

E. coli Chemically Competent Cells

1. Streak fresh E. coli strain onto LB plate and incubate overnight at 37°C

2. Inoculate a CFU into 10mL LB and incubate overnight at 37°C

3. Subculture 2mL of overnight culture into 200mL LB flask (1:100 dilution) and incubate at 37°C until OD600 of final culture is between 0.45 and 0.5 (approximately 3h)

4. Chill flask by immersion in ice bucket

Note: Perform all following steps on ice, do not allow cells to warm above 4°C

5. Spin cells down 2500g at 4°C for 10 minutes

6. Gently resuspend (by pipetting) cell pellet in 40mL ice-cold 0.1M MgCl2

7. Spin cells down 2500g at 4°C for 10 minutes

8. Gently resuspend (by pipetting) cell pellet in 40mL ice-cold 0.1M CaCl2

9. Incubate on ice for 30m

10. Spin cells down 2500g at 4°C for 10 minutes

11. Gently resuspend cell pellet in 10mL of 15% glycerol/0.1M CaCl2. Incubate on ice 2h

12. Pre-chill eppendorf tubes to -80°C in dry ice/ethanol bath.

13. Aliquot 220μL competent cells into each eppendorf tube while in dry ice/EtOH bath. Store at -80°C.