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Corynebacterium glutamicum

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.510 12/2001 Microorganism Corynebacterium glutamicum Cell type Bacteria, gram positive Molecules injected Plasmid DNA Growth medium LBG (LB with 0.5% glucose) supplemented with 2.5% glycine and isonicotinic acid hydrazid (4 mg/ml) Washing solution 15% glycerol (v/v) Electroporation solution 15% glycerol (v/v) Outgrowth medium LBG medium (LB with 0.5% glucose) Cuvette 2 mm gap width Reference Haynes J. and Britz M. 1990 Journal of General Microbiology 136 255-263 Making electrocompetent cells:

1. Grow cells in LBG medium at 30 C and shaking at 200 rpm to an O.D.600 of 0.15 to 0.25. 2. Harvest by centrifugation. 3. Wash in one culture volume of 15% glycerol. 4. Resuspend cells in 0.002 culture volumes of 15% glycerol (number of cells: approx. 2.5 x 1010 cells/ml).

Electroporation of cells:

  1. Add 1-2 l plasmid DNA (in water) to a minimum of 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) 2,500 V Time constant (T) 5 ms
  4. Immediately transfer cell suspension into LBG medium at a 1:25 dilution, incubate 1 hour at 30 C.
  5. Plate cells on selective plates; incubate for 4 days.
Expected Results: Transformation efficiency up to 5 x 105 transformants/g of DNA.



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