bruno wrote:
>> Despite all the tries I have done, I am desperatly looking for
> an efficient yeast electroporation protocol...
> I have heard that the most important thing when electroporating
> cells was to correctly find the right pulse lenght...
> Thanks to everybody who will give me some hope to believe in
> this technic which works so well with bacteria..Hi bruno,
we are currently use electroporation ofr yeast without any problem.
here follow these conditions:
taking a 3 weeks-less-old colony, growth in 100 ml Yeast Extract 1%,
bactopeptone 1% and glucose 1%. Go to 1.3-1.5OD600nm/ml (10E8cell/ml).
Centrifuge in two falcon tubes two times at 5000 rpm and wash with 100
ml of sterile, cold water, centrifuge, wash with 50 ml water,
centrifuge, suspend in 4 ml 1 M sorbitol (sterile, cold), centrifuge,
suspend in 0.1 ml of 1M sorbitol (s, c). let the yeasts at 4°C. they can
be used immediadely or one or two days after. yeasts are aliquoted in 40
µl fraction, mixed with 0.1µgDNA/0.1-5µl buffer (TE). the mix is let on
ice for 4 min. the apparatus (biorad) is setted to 1.5kV, 25 µF (gene
pulser) and 200 Ohms (pulse controler). pulse in a 2 mm chamber (time
constant is 4.5 - 5 msec). add 1 ml of 1 M cold sterile sorbitol, pour
on plate. colonies will appear in 48-72 h. I garanty this protocol!
Pierre Falson
