Multiporator / Electroporator 2510
Protocol No. 4308 915.532 01/2002
Salmonella typhimurium LT2
Bacteria, gram negative
Plasmid DNA (pBR322 in TE buffer)
10 mM HEPES, pH 7.0; 10% glycerol
SOC medium (without antibiotics)
2 mm gap width
Binotto J., et al 1991 Canadian Journal of Microbiology
Making electrocompetent cells:
Incoculate a flask of LB 1:100 with a fresh overnight
culture, grow at 37 C with shaking to an O.D.640
of 0.75. Chill
the cells in an ice-water bath for 15 min.
Harvest by centrifugation (4,000 x g, 10 min., 4 C).
Wash one time in the original culture volume with HEPES
and one time with 10% glycerol using 1/100 of the original volume.
Resuspend in 10% glycerol.
Electroporation of cells:
- Add 2 l plasmid DNA (20 pg to 20 ng) to 40 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.
Time constant (T)
- Immediately add 1 ml SOC medium and transfer to a sterile culture
tube. Incubate for 1 hour at 37 C with shaking.
- Dilute the cells in SOC medium and plate on selective LB plates.
Transformation efficiency up to 4 x 108
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. Salmonella typhimurium