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Listeria Monocytogenes

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 µg/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 µg plasmid DNA to 100 µl 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/µg of DNA.

Contact Information

In the United States:
Eppendorf North America, Inc.
102 Motor Parkway,
Hauppauge, NY 11788-5178
Tel: 800-645-3050
Fax: 516-334-7506
Web Site: http://www.eppendorfna.com/

Outside the United States:
Eppendorf AG
Barkhausenweg 1
22339 Hamburg
Germany

Customer Service: ++ 49 40 53 801-0

Fax Number: ++ 49 40 53 801-556

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