Multiporator / Electroporator 2510
Protocol No. 4308 915.531 12/2001
YEPD (1% yeast extract, 2% bactopeptone, 2% dextrose)
Ice-cold, sterile water; ice-cold 1 M sorbitol
Ice-cold 1 M sorbitol
1 M sorbitol
2 mm gap width
Dr. Robert Sclafani University of
Colorado Health Science Center Denver, CO USA
Making electrocompetent cells:
Inoculate 500 ml YEPD with an aliquot of an overnight
culture or a colony from a plate and grow to an O.D.600 of
1.3-1.5 (about 1 x 108
Harvest by centrifugation at 4,000 x g for 5 min. at
Wash in 500 ml ice-cold sterile water, centrifuge (4,000
x g, 5 min., at 4 C), repeat washing step with 250 ml water.
Resuspend in 20 ml ice-cold sorbitol and c
as above. Resuspend in 0.5 ml of 1 M sorbitol to a final volume of
1.0 to 1.5 ml, keep on ice.
Electroporation of cells:
- Add <5 l (0.1 g) DNA to 65 l of electrocompetent
cells. Homogenize by gently mixing with pipette several times.
Incubate 5 min on ice. 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 of cold sorbitol. Plate various aliquots onto
selective plates containing 1 M sorbitol.
Transformation efficiency up to 104
It is possible to scale this protocol down by starting with a culture
volume of 50 ml.
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