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is S.cerevisiae electroporation possible?...

Christian Velten cve at ifm.mh-hannover.de
Wed Jul 3 06:45:17 EST 1996


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%.


Christian

--      "Two bands or not two bands? - THAT is the question!"

Christian Velten
Institute for Molecular Biology              cve at ifm.mh-hannover.de
Medical School Hannover                      Compuserve:100736,2071
OE 5250, Konstanty-Gutschow-Straße 8         Tel.: 0511/532-5957
D-30623 Hannover, Germany                    Fax:  0511/532-4283



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