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Lactococcus Lactis

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Lactococcus lactis
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.519 – 12/2001
Microorganism Lactococcus lactis MG1363
Cell type Bacteria, gram positive
Molecules injected Plasmid DNA (pGK12)
Growth medium Complex medium with 1% glycine
Washing solution 0.5 M sucrose, 10% glycerol
Electroporation solution 0.5 M sucrose, 10% glycerol
Outgrowth medium Ice-cold complex medium with 0.5 M sucrose, 20 mM MgCl2, 2 mM CaCl2
Cuvette 1 mm gap width
Reference Dr. Horst Neve • Bundesanstalt für Milchforschung • Institut für Mikrobiologie Hermann-Weigmann Str. 1 • D-24103 Kiel • Phone +49 431 6091 • Fax +49 431 609222
Making electrocompetent cells:

1. Grow cells overnight at 30 °C to an O.D.620 of 0.7.
2. Wash twice with ice-cold washing solution.
3. Resuspend cells in 1/100 volume of electroporation solution. Keep on ice.

Electroporation of cells:

  1. Add 0.25 µg plasmid DNA (in water) to 100 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into cuvette.
  2. Wipe moisture from the cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Prokaryotes “O“
    Voltage (V) 2,000 V
    Time constant (T) 5 ms

  4. Add 1 ml ice-cold complex medium, incubate 2 hours at 30 °C.
  5. Plate diluted cells on selective chloramphenicol plates. Incubate 2 days at 30 °C.
Expected Results:
Transformation efficiency up to 1.4 x 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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