Yeast no longer transformable

[S. Tan]

Hey -

We've been doing routine yeast transformations in lab. Recently,
however, we were unable to transform plasmids into our yeast strains (e.g.
Hf7c from Clontech). We use fresh medium and new reagents, and have
tried LiAc method and Bio101 kit. Does anyone know what might be going
on? Do yeast cells lose "competency" after a long period of storage (at
-80oC)?

Thanks in advance,
Thomas

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[Aidan Weatherill]

I assume you have seen this already
http://www.umanitoba.ca/faculties/medicine/units/human_genetics/gietz/Trafo.
html

Yeast will tend to go petite (lose there mitochondria) when frozen, I am not
sure if this will affect there compatance, but you can select for whole
cells by growing on YEPEG plates.

YEPEG
1. Dissolve 10 gm of succinic acid in 500 ml of distilled H20
2. add 10 gm yeast extract
20 gm bactopeptone
20.8 ml of glycerol
3. adjust pH to 5.5 with 1 M base
4. add 20 gm agar
5. add distilled H20 to 970 ml
6. autoclave
7. when cool add 27.5 ml 95% ETOH

S. Tan wrote in message <808abj$e...@net.bio.net>...

[Mark Hartmann]

On 8 Nov 1999 21:10:11 -0800, S. Tan <sl...@u.washington.edu> wrote:

>Hey -
>
>We've been doing routine yeast transformations in lab. Recently,
>however, we were unable to transform plasmids into our yeast strains (e.g.
>Hf7c from Clontech). We use fresh medium and new reagents, and have
>tried LiAc method and Bio101 kit. Does anyone know what might be going
>on? Do yeast cells lose "competency" after a long period of storage (at
>-80oC)?

In fact yeast cells lose their competence when you store it at -80°C
for a longer time period. I would never use such yeast cells for
transformation of a CDNA library for example. I allways would use
freshly prepaired cells for high efficiency transformations.

Regards
Mark

[Lutz Thon]

Hi,
look at this site. There is very much information!
http://www.umanitoba.ca/faculties/medicine/units/human_genetics/gietz/Trafo.html

Bye
Lutz