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:
- 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.
- Wipe moisture from the cuvette and insert the cuvette into the device.
- Electroporation:
Mode
Prokaryotes O
Voltage (V)
1,000 V
Time constant (T)
5 ms
- Immediately add 1 ml of BHI medium and incubate at 37 C for one
hour.
- Plate cells on selective BHI plates.
Expected Results:
Transformation efficiency up to 4 x 10
6 transformants/g
of DNA.
'"/>Source:
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