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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.

Electr oporation of cells:

  1. Add up to 5 g plasmid DNA (1 g/l) 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) 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
  5. Plate on selective LB plates.
Expected Results: Transformation efficiency up to 107 transformants/g of DNA.



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