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Corynebacterium Glutamicum

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Corynebacterium glutamicum
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.510 – 12/2001
Microorganism Corynebacterium glutamicum
Cell type Bacteria, gram positive
Molecules injected Plasmid DNA
Growth medium LBG (LB with 0.5% glucose) supplemented with 2.5% glycine and isonicotinic acid hydrazid (4 mg/ml)
Washing solution 15% glycerol (v/v)
Electroporation solution 15% glycerol (v/v)
Outgrowth medium LBG medium (LB with 0.5% glucose)
Cuvette 2 mm gap width
Reference Haynes J. and Britz M. •1990 • Journal of General Microbiology 136 • 255-263
Making electrocompetent cells:

1. Grow cells in LBG medium at 30 °C and shaking at 200 rpm to an O.D.600 of 0.15 to 0.25.
2. Harvest by centrifugation.
3. Wash in one culture volume of 15% glycerol.
4. Resuspend cells in 0.002 culture volumes of 15% glycerol (number of cells: approx. 2.5 x 1010 cells/ml).

Electroporation of cells:

  1. Add 1-2 µl plasmid DNA (in water) to a minimum of 40 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. Immediately transfer cell suspension into LBG medium at a 1:25 dilution, incubate 1 hour at 30 °C.
  5. Plate cells on selective plates; incubate for 4 days.
Expected Results:
Transformation efficiency up to 5 x 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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