Electrocompetent E. coli Cells (preliminary protocol)

(This protocol works, I just don’t know the transformation efficiencies at this time)

Gregory W. Broussard

Revised:  5/31/12

(Please use, change and disseminate freely!)

This protocol is for a 100 mL culture and will yield ~40, 100 mL aliquots.

1. Inoculate two 5 mL cultures of E. coli NEB5alpha cells in LB broth and grow overnight.
2. Subculture into a 500 mL flasks (nonbaffled) by adding 3 ml of one of the culture from #1 into 100 mL of the same media.  Check O.D.₆₀₀ and incubate shaking at 37° C for 2-4 hours.
3. When cells reach an OD₆₀₀ of ~0.5, transfer the cells to two 50 mL conical tubes.  Incubate the tubes on ice for 30 min to 1 hr.
4. Centrifuge the cells at 5000 rpm for 10 min and discard the supernatant.
5. Wash the cells in 1/2 vol. (25 mL) 10% glycerol and centrifuge as above.
6. Wash the cells in 1/4 vol. (12.5 mL) 10% glycerol and centrifuge as above.
7. Wash the cells in 1/8 vol. (6.25 mL) 10% glycerol and centrifuge as above.
8. Suspend the cells in 2 mL 10% glycerol.
9. Separate into 100 mL aliquots in microcentrifuge tubes.
10. Flash freeze on dry ice and store at -80° C or use immediately.