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Saccharomyces Cerevisiae

Saccharomyces cerevisiae
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.531 – 12/2001
Microorganism Saccharomyces cerevisiae
Cell type Yeast
Molecules injected Plasmid DNA
Growth medium YEPD (1% yeast extract, 2% bactopeptone, 2% dextrose)
Washing solution Ice-cold, sterile water; ice-cold 1 M sorbitol
Electroporation solution Ice-cold 1 M sorbitol
Outgrowth medium 1 M sorbitol
Cuvette 2 mm gap width
Reference Dr. Robert Sclafani • University of Colorado • Health Science Center • Denver, CO • USA
Making electrocompetent cells:

1. Inoculate 500 ml YEPD with an aliquot of an overnight culture or a colony from a plate and grow to an O.D.600 of 1.3-1.5 (about 1 x 108 cells/ml).
2. Harvest by centrifugation at 4,000 x g for 5 min. at 4 °C.
3. Wash in 500 ml ice-cold sterile water, centrifuge (4,000 x g, 5 min., at 4 °C), repeat washing step with 250 ml water.
4. Resuspend in 20 ml ice-cold sorbitol and centrifuge as above. Resuspend in 0.5 ml of 1 M sorbitol to a final volume of 1.0 to 1.5 ml, keep on ice.

Electroporation of cells:

  1. Add <5 µl (0.1 µg) DNA to 65 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Incubate 5 min on ice. 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) 1,500 V
    Time constant (T) 5 ms

  4. Immediately add 1 ml of cold sorbitol. Plate various aliquots onto selective plates containing 1 M sorbitol.
Expected results:
Transformation efficiency up to 104 to 105 transformants/µg of DNA.
Note:
It is possible to scale this protocol down by starting with a culture volume of 50 ml.

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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