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Kluyveromyces Lactis

Kluyveromyces lactis
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.516 – 12/2001
Microorganism Kluyveromyces lactis AWJ137
Cell type Yeast
Molecules injected DNA
Growth medium Not given
Washing solution YPD, 10 mM DTT, 40 mM HEPES (pH 8.0)
Electroporation solution 2.5 mM Tris/HCl, 1 mM MgCl2, (pH 8.0)
Outgrowth medium YPD with 1 M sorbitol
Cuvette 4 mm gap width
Reference Prof. Friedhelm Meinhardt • Universität Münster • Corrensstr. 3 • D-48149 Münster
e-mail: meinhar@uni-muenster.de
Making electrocompetent cells:

1. Grow a 50 ml cell culture of Kluyveromyces lactis to a density of 5 x 107 cells/ml.
2. Harvest by centrifugation (5 min., at 4,000 x g)
3. Resuspend cells in 5 ml washing solution and incubate for 5 min.
4. Wash twice with electroporation solution and resuspend in 0.25 ml of the same solution. Incubate for 1 hour on ice.

Electroporation of cells:

  1. Add 5 µl DNA (1 µg/µl) 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. Add 1 ml YPD medium with 1 M sorbitol, incubate for 1 hour at 30 °C.
  5. Centrifuge cells 30 sec., resuspend in 0.4 ml sterile water, plate in aliquots on selective plates.
Expected results:
Transformation efficiency up to 200 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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