McKinney:TransformByElectroporation

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[[Category:Protocol]]
[[Category:Protocol]]
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[[Category:In vivo]]
 
[[Category:Mycobacterium smegmatis]]
[[Category:Mycobacterium smegmatis]]

Current revision

Materials

  • 50mL 7H9 mycobacterial medium + 3mL per transformation
  • 102mL 10% glycerol (possibly a few mL more if you are doing many transformations)
  • 400mL PBS + 0.1% Tween 80 per transformation
  • electroporation cuvettes and electroporator
  • LB agar plates carrying the relevant antibiotic markers
  • frozen aliquot of M. smegmatis

Directions

  1. Thaw the aliquot of M. smegmatis and inoculate 50mL 7H9 with 100-200μL of it (depending on concentration of stock). Incubate shaking at 37°C until the optical density at 600nm is between 0.6 and 0.8. This is the range where you get the highest transformation efficiency.
  2. Divide the culture into two 25mL screw cap tubes. Pellet the cells at 3,000rpm for 10 minutes and resuspend each in 25mL glycerol. Repeat twice more, the last time resuspending each in 1mL glycerol. This ensures that the medium is fairly pure. If it's not, then the medium will arc when you electroporate.
  3. Prepare the recovery tubes: one test tube of 3mL 7H9 per transformation. Put 300μL of cells in 10% glycerol in each electroporation cuvette, add the DNA (on the order of 3ng per transformation).
  4. Electroporate at 2.5kV, 1kΩ, and 25μF. Should yield time constants of 15 to 25ms. Immediately transfer the cells to their recovery tube. When all the transformations are done, incubate the recovery tubes shaking at 37°C for at least 3 hours.
  5. Pellet the tubes at 3,000 rpm for 10 minutes, and resuspend in 400μL PBS + 0.1% Tween 80. Plate each tube on two LB agar plates with the relevant antibiotics, 200μL per plate. Incubate the plates at 37°C.

Notes

Unlike E. coli, there is no need to keep mycobacteria cold when transforming them. Indeed, there is some evidence that M. tuberculosis may transform better at room temperature.

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