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Pseudomonas Aeruginosa

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Pseudomonas aeruginosa
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.528 – 01/2002
Microorganism Pseudomonas aeruginosa
Cell type Bacteria, gram negative
Molecules injected Plasmid DNA (pUC 18 with 1.8 kb insert in water)
Growth medium LB medium
Washing solution 300 mM sucrose
Electroporation solution 300 mM sucrose
Outgrowth medium LB medium
Cuvette 2 mm gap width
Reference Smith A.W. and Iglewski B.H. • 1989 • Nucleic Acids Research 17, No. 24 • 10509
Making electrocompetent cells:

1. Grow cells in LB medium at 37 °C with shaking up to an O.D.540 of 0.3-05.
2. Harvest by centrifugation (7,000 x g, 10 min, at 4 °C).
3. Wash pellet in the original volume with sucrose, centrifuge and wash again in _ volume of washing solution.
4. Resuspend in 300 mM sucrose to a final concentration of 1011 cells/ml, chill on ice for 30 minutes.

Electroporation of cells:

  1. Add up to 5 µg plasmid DNA (1 µg/µl) to 40 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) 1,600 V
    Time constant (T) 5 ms

  4. Add 1 ml LB medium, transfer to a sterile tube containing additional 2 ml of LB. Incubate 2 hours at 37 °C with
    shaking.
  5. Plate on selective LB plates.
Expected Results:
Transformation efficiency up to 107 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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