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Mycobacterium Intracellulare

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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:

  1. Add 1 µg plasmid DNA to 100 µl 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/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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