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Mycobacterium Smegmatis

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Mycobacterium smegmatis
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.522 – 03/2002
Microorganism Mycobacterium smegmatis LR222
Cell type Bacteria, gram positive
Molecules injected Plasmid DNA
Growth medium Middlebrook 7H9 medium with 0.1% Tween and Dubos oleic albumin complex
enrichment
Washing solution 10% glycerol
Electroporation solution 10% glycerol
Outgrowth medium Middlebrook 7H9 liquid medium with 0.1% Tween and Dubos oleic albumin complex
enrichment, selective Middlebrook 7H10 agar plates
Cuvette 1 mm gap width
Reference Beggs, M. L.. et al • 1995 • Journal of Bacteriology 177, No. 17 • 4836-4840
Making electrocompetent cells:

1. Grow cells in sidearm flask or tissue culture plates with gentle agitation until a Klett unit reading of 100 to 200 was
obtained.
2. Harvest cells and wash three times with cold sterile 10% glycerol.
3. Resuspend 1/100 volume of 10% glycerol.

Electroporation of cells:

  1. Add up to 1 µg plasmid DNA to 60 µl of electrocompetent cells. Homogenize by gently mixing with pipette several
    times. Incubate on ice for 10 min. 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. Wash cells out of the cuvette with 1 ml of outgrowth medium and incubate for 2 h at 37 °C.
  5. Plate on selective agar.
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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