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Pseudomonas Putida

Pseudomonas putida
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.529 – 01/2002
Microorganism Pseudomonas putida P8
Cell type Bacteria, gram negative
Molecules injected Plasmid DNA
Growth medium Standard 1-medium
Washing solution 10% glycerol
Electroporation solution 10% glycerol
Outgrowth medium Standard 1-medium
Cuvette 2 mm gap width
Reference Prof. Friedhelm Meinhardt • Universität Münster • Corrensstraße 3 • D-48149 Münster
e-mail: meinhar@uni-muenster.de
Making electrocompetent cells:

1. Inoculate 50 ml standard-1 medium with 7 ml of a fresh overnight culture of Pseudomonas putida. Grow cells at 30 °C to a density of O.D. of 0.8.
2. Harvest by centrifugation.
3. Wash twice with 50 ml ice-cold glycerol, centrifuge.
4. Resuspend cells in 0.8 ml ice-cold glycerol, keep on ice.

Electroporation of cells:

  1. Add 4 µl plasmid DNA (1 µg) 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) 2,400 V
    Time constant (T) 5 ms

  4. Immediately add 1 ml standard 1-medium. Incubate 2 hours at 30 °C.
  5. Plate cells on selective plates.
Expected Results:
Transformation efficiency up to 2 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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