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Mycobacterium Tuberculosis

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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 µg plasmid DNA to 100 µl (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/µg of DNA.

Contact Information

In the United States:
Eppendorf North America, Inc.
102 Motor Parkway,
Hauppauge, NY 11788-5178
Tel: 800-645-3050
Fax: 516-334-7506
Web Site: http://www.eppendorfna.com/

Outside the United States:
Eppendorf AG
Barkhausenweg 1
22339 Hamburg
Germany

Customer Service: ++ 49 40 53 801-0

Fax Number: ++ 49 40 53 801-556

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