Multiporator / Electroporator 2510
Protocol No. 4308 915.524 03/2002
Mycobacterium tuberculosis H37Rv
Bacteria, gram positive
Middlebrook 7H9 medium with 0.2% glycerol, 0.25% Tween
80, albumin-dextrose complex (ADC)
Middlebrook 7H9 medium with ADC, selective 7H10 agar
plates with ADC
2 mm gap width
Armitige, L. Y. et al 2000
Infection and Immunity 68, No. 2 767-778
Making electrocompetent cells:
Grow cells with gentle shaking to an OD600
Harvest cells and wash three times in 1/50 volume of
cold 10% glycerol.
Resuspend in 10% glycerol at a concentration of approx.
cells/ml and store at 70 C until needed.
Electroporation of cells:
- 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.
- Wipe moisture from the cuvette and insert the cuvette into the device.
Time constant (T)
- Immediately add 1 ml outgrowth medium and incubate at 37 C for
2.5 h with agitation.
- Plate on selective agar plates and incubate at 37 C for 3 weeks
under 9.3% CO2.
Transformation efficiency up to 105
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. Mycobacterium intracellulare3
. Mycobacterium smegmatis4
. Mycobacterium avium5
. Mycobacterium intracellulare6
. Mycobacterium smegmatis7
. Mycobacterium tuberculosis