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Staphylococcus Aureus

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Staphylococcus aureus
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.533 – 01/2002
Microorganism Staphylococcus aureus
Cell type Bacteria, gram positive
Molecules injected Plasmid DNA (pC194)
Growth medium Basic medium (BM: 1% peptone, 0.5% yeast extract, 0.1% glucose, 0.5% NaCl,
0.1% K2HPO4)
Washing solution 10% glycerol (in distilled water)
Electroporation solution 10% glycerol (in distilled water)
Outgrowth medium SMMP50 medium (5.5 parts SMM buffer (1 M sucrose, 0.04 M maleic acid, 0.04 M MgCl2, pH 6.5), 4 parts 7% Panassy broth, 0.5 parts 10% BSA)
Cuvette 2 mm gap width
Reference Augustin J. and Götz F. • 1990 • FEMS Microbiology Letters 66 • 203-208
Making electrocompetent cells:

1. Inoculate a flask of BM with a fresh overnight culture. Grow at 37 °C with shaking to an O.D.578 of about 0.5 to 0.65.
2. Wash in one volume of 10% glycerol at room temperature, followed by washes with 1/2, 1/20 and 1/50 volumes.
3. Resuspend to final volume of 1-5 x 1010 cells/ml in 10% glycerol. Aliquote and freeze at –70 °C.

Electroporation of cells:

  1. Thaw cells for 5 min at room temperature. Add 1-2 µl plasmid DNA (100-500 ng) to 50 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Incubate at room temperature for 30 minutes.
  2. Transfer mixture into a cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Prokaryotes “O“
    Voltage (V) 1,800 V
    Time constant (T) 5 ms

  4. Add 1 ml SMMP50 medium to the cells and transfer to sterile tube. Incubate 90 minutes at 37 °C with moderate shaking.
  5. Plate on selective BM plates. Incubate 20 hours at 37 °C.
Expected Results:
Transformation efficiency up to 1.5 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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