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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 Universitt Mnster Corrensstrae 3 D-48149 Mnster
e-mail: 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 l plasmid DNA (1 g) to 40 l 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/g of DNA.



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