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Nocardia Asteroides

Nocardia asteroides
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.525 – 01/2002
Microorganism Nocardia asteroides
Cell type Bacteria, gram positive
Molecules injected Plasmid DNA (in TE buffer)
Growth medium N2 medium (20 g/l glucose, 2 g/l yeast extract, 4 g/l beef extract, 6 g/l tryptone, 2 g/l NaCl, 10 g/l glycine)
Washing solution Distilled water
Electroporation solution 0.3 M sucrose, 15% glycerol
Outgrowth medium N2 medium (without antibiotics)
Cuvette 2 mm gap width
Reference Yao W., et al • 1994 • Current Microbiology 29 • 223-227
Making electrocompetent cells:

1. Inoculate a fresh overnight culture of bacteria into N2 medium. Grow cells at 28 °C with shaking to an O.D.600 of 0.7.
2. Harvest by centrifugation.
3. Wash twice with _ volume of distilled water.
4. Resuspend in a final volume of 1/20 of the original volume of electroporation solution.

Electroporation of cells:

  1. Add 2 µl (0.1 µg) plasmid DNA to 50 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 N2 medium and incubate for 2 hours at 30 °C.
  5. Plate cells on selective YEME medium with a 3 ml YEME soft agar (0.4%) overlayment.
Expected Results:
Transformation efficiency up to 8 x 104 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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