In article <31D95815.30C3 at verlaine.saclay.cea.fr>, Falson
<falson at verlaine.saclay.cea.fr> wrote:
> 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
You can increase the transformation rate by putting the cells after first
collection for 45 min into LiAc/TE (20 ml/100 ml culture) and adding DTT
(500 microl 1M DTT/20 ml) for another 15 min. Then ...
> 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!
You can also freeze the final Sorbitol-suspension. Max. decrease of
transformation rate about 10%.
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