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.


'"/>

Source:


Page: All 1 2
Related biology technology :

1. Listeria monocytogenes
2. Two-dimensional Optimization of a Semi-nested PCR for Detecting Listeria monocytogenes
3. Use of the DCode System to Detect the Food-Borne Bacterial Pathogen Listeria monocytogenes