Quick Electrocompetent M. smegmatis

Gregory Broussard

Revised: 11/24/08

(Please use, change and disseminate freely!)

Disclaimer: This protocol DOES NOT give efficient electroporations. It will give you 10-30 colonies with 1 µl of prepped plasmid. ONLY use with supercoiled DNA when you need to get stuff done TODAY!!!

1. Centrifuge 1.5 ml culture in a microfuge tube at 5,000 X g for 1 minute.
2. Resuspend pellet in 1 ml cold 10% glycerol. (keep on ice when not centrifuging) Centrifuge again.
3. Repeat step #2 twice more for a total of three washes with cold 10% glycerol.
4. After the final wash suspend cells in 100 µl cold 10% glycerol and place on ice.
5. Add 1 µl or desired amount of plasmid DNA and sit on ice for 10 min.
6. Electroporate at 2.5kV, 1000 Ω, 25 µF.
7. Add 1 ml 7H9/ADC/Tw to cells.
8. Recover for 2 hrs. at 37 ºC, shaking.
9. Plate with appropriate antibiotics.