e for 15 minutes and transfer to a
prechilled centrifuge bottle. Harvest by centrifugation (20 minutes,
5,000 x g, 2-4 C). Resuspend pellet in 5 ml ice-cold water. Keep
the cells cold during the entire procedure.
Wash twice with the original culture volume of ice-cold
water. Centrifuge as above. Resuspend pellet by swirling in
If using the cells immediately, place suspension in
a prechilled tube and centrifuge (10 minutes, 5,000 x g, 2-4 C).
Resuspend the cells in ice-cold water to a final concentration of
approximately 2 x 1011
cells/ml. Aliquote 40-300 l
cells into prechilled centrifuge tubes.
If freezing the cells for later use, add 40 ml of ice-cold
10% glycerol, mix and centrifuge for 10 minutes, at 5,000 x g and
2-4 C. Resuspend the cells in ice-cold 10% glycerol to a final
concentration of approximately, 2 x 1011
40-300 l cells into prechilled centrifuge tubes. quick freeze
on dry ice and store at 80 C.
Electroporation of cells:
- Add 1 l DNA (10 pg in water) to tubes containing 40 l
electrocompetent cells. Homogenize by gently mixing with
pipette several times.
- Transfer mixture to a prechilled cuvette. Wipe moisture from the cuvette
and insert the cuvette into the device.
Source:Page: All 1 2 3 Related biology technology :1
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