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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 Kornyi, 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 l aliquots in liquid nitrogen and store at 70 C.

Electroporation of cells:

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

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

  4. Electroporation:

  5. Mode Prokaryotes O Voltage (V) 1,800 V Time constant (T)
    5 ms No. of pulses (n) 1
  6. Immediately add 1 ml AG medium and incubate with shaking at 200 rpm for 1 h at 30 C.

  7. Plate onto selective AG media plates.

Expected results:

Transformation efficiency up to 6.8 x 105 transformants/g of DNA.



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