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Listeria monocytogenes

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.520 12/2001 Microorganism Listeria monocytogenes 23074 Cell type Bacteria, gram positive Molecules injected Plasmid DNA Growth medium BHI, supplemented with 0.5 M sucrose Washing solution 1 mM HEPES (pH 7.0), 0.5 M sucrose Electroporation solution 1 mM HEPES (pH 7.0), 0.5 M sucrose Outgrowth medium BHI, supplemented with 0.5 M sucrose Cuvette 1 mm gap width Reference Park, S. and Stewart, G. 1990 Gene 94 129-132 Making electrocompetent cells:

1. Dilute an overnight culture of L. monocytogenes 23074 in BHI, into fresh media (1:100). Grow at 37 C with shaking until reaching an O.D.600 of 0.2. 2. Add Penicillin G (10 g/ml) and continue incubation for a further 2 hours. 3. Harvest by centrifugation (8,000 x g, 10 min., at 4 C) and wash three times in ice-cold washing solution, once with equal volume and twice with _ volume. 4. Resuspend the cells in ice-cold electroporation solution (0.0025 vol.). Use cells within 30 minutes of their preparation.

Electroporation of cells:

  1. Add 1 g plasmid DNA to 100 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) 1,000 V Time constant (T) 5 ms
  4. Immediately add 1 ml of BHI medium and incubate at 37 C for one hour.
  5. Plate cells on selective BHI plates.
Expected Results: Transformation efficiency up to 4 x 106 transformants/g of DNA.



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