Multiporator / Electroporator 2510
Protocol No. 4308 915.526 01/2002
Bacteria, gram positive
Plasmid DNA (in TE buffer)
N2 medium (20 g/l glucose, 2 g/l yeast extract, 4 g/l
beef extract, 6 g/l tryptone, 2 g/l NaCl, 10 g/l glycine)
0.3 M sucrose, 15% glycerol
N2 medium (without antibiotics)
2 mm gap width
Yao W., et al 1994 Current
Microbiology 29 223-227
Making electrocompetent cells:
Inoculate a fresh overnight culture of bacteria into
N2 medium. Grow cells at 28 C to an O.D.600
Harvest by centrifugation..
Wash twice with _ volume of distilled water.
Resuspend in a final volume of 1/20 of the original
volume of electroporation solution.
Electroporation of cells:
- Add 2 l (0.1 g) plasmid DNA to 50 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)
- Add 1 ml N2 medium and incubate for 2 hours at 30 C.
- Plate cells on selective YEME medium with a 3 ml YEME soft agar (0.4%)
Transformation efficiency up to 2.8 x 103
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