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Mycobacterium intracellulare

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.521 02/2002 Microorganism Mycobacterium intracellulare 1403 Cell type Bacteria, gram positive Molecules injected Plasmid DNA Growth medium Middlebrook 7H9 liquid medium with oleic acid dextrose complex (OADC) Washing solution 10% glycerol Electroporation solution 10% glycerol Outgrowth medium Middlebrook 7H9 liquid medium with oleic acid dextrose complex Cuvette 2 mm gap width Reference Marklund, B.-I. et al 1995 Journal of Bacteriology 177, No. 21 6100-6105 Making electrocompetent cells:

1. Grow a 50-100 ml cell culture at 37 C statically to an O.D.600 of 0.1 to 0.3. 2. Harvest by centrifugation. 3. Wash twice in cold 10% glycerol. The first time in 30 ml the second time in 1 ml. 4. Resuspend 0.2-0.5 ml of 10% glycerol.

Electroporation of cells:< /b>

  1. Add 1 g plasmid DNA to 100 l of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.
  2. Wipe moisture from the cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Prokaryotes O Voltage (V) 2,500 V Time constant (T) 5 ms
  4. Transfer cells to 1 ml of 7H9 medium and grow statically for 16-20 h.
  5. To disperse cells, bath sonicate cultures twice for 30 s each time. Dilute suspension in 0.9% NaCl supplemented with 0.1% Tween 80.
  6. Plate on selective agar.
Expected Results: Transformation efficiency up to 106 transformants/g of DNA.



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