Electrocompetent Mycobacterium Cells

Gregory W. Broussard

Revised: 01/20/09

(Please use, change and disseminate freely!)

This procedure is used for M. smegmatis and M. tuberculosis electrocompetent cell preparations. M. smeg cells should be kept cold by using ice-cold glycerol and centrifuging at 4° C. M. tb cells and glycerol should be kept warm and centrifugations should be performed at RT. These guidelines will improve transformation efficiency. This protocol is for a 50 mL culture and will yield ~20, 100 µL aliquots.

1. Inoculate a 3 mL culture of M. smeg mc2 155 in 7H9 broth (containing ADC, CB, CHX, Tw) and grow until saturation (~2 days).
2. Subculture into 50 mL of the same media in a 250 mL flask (nonbaffled) to a final OD₆₀₀ = 0.020 and grow shaking at 37° C overnight.
3. On the following day, when cells reach an OD₆₀₀ of 0.8-1.0, transfer the cells to 50 mL conical tubes. For M. smeg, incubate the tubes on ice for 30 min. to 2 hrs.
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 3 mL 10% glycerol.
9. Separate into 100 µL aliquots in microcentrifuge tubes.
10. Freeze on dry ice and store at -80° C or use immediately.

Notes:

1. Using M. tb immediately will improve efficiency.
2. If arching occurs after thawing cells, do two mini washes in 1 mL 10% glycerol and resuspend in 100 µL for electroporation.