bacillus subtilis electroporation

[Susanne Rohrer]

"R. Jayakumar" wrote:

> Can anyone suggest a good procedure and what are the
> electroporation conditions for doing it.  I have a genepulser II aparatus
> with me with a pulsecontroller II module attached with it.

I can't say for sure for B. subtilis, but with S. aureus we do the following:
Grow up cells to OD 0.6
Wash 4x successively in 0.5, 0.25, 0.125, and 0.0625 times the original culture
volume of ice-cold, sterile, 0.5M sucrose
at one of these steps, incubate 2-4 hrs on ice. We don't know why, but I have a
feeling it might be to elute all the salt from the cell wall (?)
resuspend cells in 10% glycerol, 4ml per liter original culture volume
snap freeze in LN2 in 50ul aliquots

TF:
thaw on ice
incubate with DNA 30 min on ice
Electroporate at 1.5kV, 25uFd, 100 Ohm in 0.1cm cuvettes
add 900ul SOC
recover for 90min before plating

good luck

Susanne