In article <31D95815.30C3 at verlaine.saclay.cea.fr>, Falson
<falson at verlaine.saclay.cea.fr> wrote:
>..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
We do the same thing, with the following modifications:
1) Use a 5ml overnight grown in YPD. Make it from a fresh colony. Wash
the cells 3x in 5 ml water, 2x in 1M sorbitol, and resuspend the pellet of
cells in whatever 1M sorbitol remains after pouring off the second wash.
2) We have found that the addition of carrier DNA (12.5
ugm/transformation) enhances the yield of integrative transformants about
10x. See Schiestl and Gietz (Curr Genet 1989 Dec;16(5-6):339-46) for how
to make carrier.
3) We find that plating at lower cell concentrations often helps recover
transformants. If you put too many cells on a single plate, they'll eat
up all the goodies.
Good luck!
--
Michael Lichten
lichten at helix.nih.gov
