Madhadron:ElectroporateSmegmatis

Materials

• Sterile water with 0.05% tween-80
• 7H9 medium
• LB plates with: no marker; 50μg/mL hygromycin and 25μg/mL kanamycin; 25μg/mL kanamycin

Directions

1. Grow 100mL culture of M. smegmatis overnight to A₆₀₀ = 0.6-0.8.
2. Centrifuge culture at 3,000rpm for 10 minutes at 4°C. Aspirate supernatant and resuspend in same volume of sterile water + 0.05% tween-80. Repeat.
3. Centrifuge once more, and resuspend in 2mL of just sterile water (no tween).
4. Electroporate 400μL plus DNA with 2.5kV, 1kΩ 25μF, and immediately transfer electroporated slurry to 5mL recovery medium.
5. Incubate recovery medium shaking at 37°C for at least 3.5 hours.
6. Centrifuge recovery medium at 3,000 rpm for 10 minutes. Resuspend in 300μL PBS + 0.05% tween-80 and plate out, 100μL per plate, on blank (no antibiotics), kanamycin, and hyg+kan.

Notes

• Positive controls: an integrating plasmid (pMV361); no DNA with electroporation on plain LB; no DNA without electroporation on plain LB.
• Negative controls: no DNA with and without electroporation on antibiotic plates.