Electroporation of Mycobacteria

Gregory W. Broussard

Revised: 10/22/07

(Please use, change and disseminate freely!)

This transformation procedure assumes the use of a BioRad pulse-controller electroporation unit. If using fresh M. tb cells, without freezing, the cells are not incubated on ice after adding DNA.

1. Thaw tubes of cells on ice for ~10 min.
2. Add DNA (1-50 ng, 100ng for linear targeting substrate DNA) to cells and mix gently. Incubate for 10 min. (on ice for M. smeg).
3. Transfer cells to a cuvet (pre-chilled for M. smeg).
4. Wipe all moisture off of the cuvet and place in electroporator holder. Electroporate with the pulse controller set to 2.5 kV, 1000 Ω, 25 μF. Check time constant, it should be between 18-22. If the cells arc, repeat the transformation (possibly do further washes with 10% glycerol).
5. For recovery, 1 mL 7H9 broth (containing ADC and Tw, and oleic acid if for M. tb) is added to the cells and cells are transferred to a test tube and incubated at 37° C. For plasmid DNA in M. smeg, recover for 2 hrs., in M. tb recover overnight. For transformations with linear targeting substrate DNA recover for 4hrs. in M. smeg and for 72 hrs. in M. tb.
6. Plate on selective media and incubate at 37° C.