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Mycobacterium tuberculosis

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.524 03/2002 Microorganism Mycobacterium tuberculosis H37Rv Cell type Bacteria, gram positive Molecules injected Plasmid DNA Growth medium Middlebrook 7H9 medium with 0.2% glycerol, 0.25% Tween 80, albumin-dextrose complex (ADC) Washing solution 10% glycerol Electroporation solution 10% glycerol Outgrowth medium Middlebrook 7H9 medium with ADC, selective 7H10 agar plates with ADC Cuvette 2 mm gap width Reference Armitige, L. Y. et al 2000 Infection and Immunity 68, No. 2 767-778 Making electrocompetent cells:

1. Grow cells with gentle shaking to an OD600 of 0.6-1.0. 2. Harvest cells and wash three times in 1/50 volume of cold 10% glycerol. 3. Resuspend in 10% glycerol at a concentration of approx. 1011 cells/ml and store at 70 C until needed.

Electroporation of cells:

  1. Add 2-4 g plasmid DNA to 100 l (1010) 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) 1,200 V Time constant (T) 5 ms
  4. Immediately add 1 ml outgrowth medium and incubate at 37 C for 2.5 h with agitation.
  5. Plate on selective agar plates and incubate at 37 C for 3 weeks under 9.3% CO2.
Expected Results: Transformation efficiency up to 105 transformants/g of DNA.



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