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Azotobacter Vinelandii

Azotobacter vinelandii
Multiporator / Electroporator 2510
Transformation Protocol

Protocol No. 4308 915.503 – 04/2002
Microorganism Azotobacter vinelandii
Cell type Bacteria, gram negative
Molecules injected Plasmid DNA
Growth medium Nitrogen-free medium
Washing solution Ice-cold 10% sterile glycerol
Electroporation solution Ice-cold 10% sterile glycerol
Outgrowth medium Azotobacter growth (AG) medium
Cuvette 1 mm gap width
Reference Korányi, P. et al • 1998 • Research in Microbiology 149 • 361-372

Making electrocompetent cells:

  1. Cultivate cells with vigorous shaking at 30 °C to an O.D.620 of 0.4-0.5.

  2. Harvest by centrifugation at 5,000 rpm for 10 min at 0 °C.

  3. Wash with the original culture volume of ice-cold 10% glycerol.

  4. Repeat this step three times using a half and a quarter of the original volume, and finally 2-4 ml of the glycerol solution (7.0 x 107 cells/ml final cell concentration).

  5. Freeze 40 µl aliquots in liquid nitrogen and store at –70 C.

Electroporation of cells:

  1. Add 1 µl (300 ng/µl) plasmid DNA to 40 µl of electrocompetent cells. Homogenize by gently mixing with pipette

    2. several times. Transfer mixture into a prechilled cuvette.
  2. Wipe moisture from the cuvette and insert the cuvette into the device.

  3. Electroporation:

    4. Mode Prokaryotes “O“
    Voltage (V) 1,800 V
    Time constant (T)
    		 5 ms
    No. of pulses (n) 1

  4. Immediately add 1 ml AG medium and incubate with shaking at 200 rpm for 1 h at 30 °C.

  5. Plate onto selective AG media plates.

Expected results:

Transformation efficiency up to 6.8 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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