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Clostridium Botulinum

Clostridium botulinum
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.509 – 12/2001
Microorganism Clostridium botulinum
Cell type Bacteria, gram positive
Molecules injected Plasmid DNA (pGK12 in water)
Growth medium TPGY medium
Washing solution 10% PEG 8000
Electroporation solution 10% PEG 8000
Outgrowth medium TPGY medium with 25 mM MgCl2
Cuvette 4 mm gap width
Reference Zhou Y. and Johnson E. A. • 1993 • Biotechnology Letters • 15, No. 2 • 121-126
Making electrocompetent cells:

1. Grow cells in TPGY medium at 37 °C to an O.D.660 of 0.8.
2. Harvest by centrifugation. Wash in 0.8 volumes of PEG at 4 °C, keep cells cold during the entire procedure.
3. Resuspend cells in 0.8 ml 10% PEG (number of cells: 4 x 108 cells/ml), keep on ice.

Electroporation of cells:

  1. Add 1 µg plasmid DNA to 800 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette. Keep on ice for 2 min.
  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. Dilute cells into 10 ml TPGY medium with 25 mM MgCl2 and incubate 5 hours at 34 °C.
  5. Plate cells on selective TPGY agar plates; incubate at 34 °C for 2-3 days.
Expected Results:
Transformation efficiency up to 3 x 103 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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