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Nocardia asteroides

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.525 01/2002 Microorganism Nocardia asteroides Cell type Bacteria, gram positive Molecules injected Plasmid DNA (in TE buffer) Growth medium N2 medium (20 g/l glucose, 2 g/l yeast extract, 4 g/l beef extract, 6 g/l tryptone, 2 g/l NaCl, 10 g/l glycine) Washing solution Distilled water Electroporation solution 0.3 M sucrose, 15% glycerol Outgrowth medium N2 medium (without antibiotics) Cuvette 2 mm gap width Reference Yao W., et al 1994 Current Microbiology 29 223-227 Making electrocompetent cells:

1. Inoculate a fresh overnight culture of bacteria into N2 medium. Grow cells at 28 C with shaking to an O.D.600 of 0.7. 2. Harvest by centrifugation. 3. Wash twice with _ volume of distilled water. 4. Resuspend in a final volume of 1/20 of the original volume of electroporation solution.

Electroporation of cells:

  1. Add 2 l (0.1 g) plasmid DNA to 50 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. Add 1 ml N2 medium and incubate for 2 hours at 30 C.
  5. Plate cells on selective YEME medium with a 3 ml YEME soft agar (0.4%) overlayment.
Expected Results: Transformation efficiency up to 8 x 104 transformants/g of DNA.



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