Need help with transformation of a library into Y190

Frederik Boernke boernke at ipk-gatersleben.de
Wed Dec 17 11:42:29 EST 1997


Snow, Sheila : NSI RAss wrote:
> 
> Hi,
> 
>     I have searched part of the archives and can't find help! I am trying to
> transform a library into Y190. I have tried sequential and simultaneous via
> electroporation to no avail. Does anyone have any suggestions!
> Thanks for any advice/experiences that you have had!
> Sheila
> snows at lhs.org
> 
> **
> ** Note from Moderator:
> **
> ** dear users: please include a title on your messages
> ** I concocted this one, appologies to Sheila if it's not
> ** exactly what she wanted ...
> **                                   francis at ncbi.nlm.nih.gov
> **

Hi Sheila,

to me electroporation seems not to be the proper way for transforming
a two-hybrid library. Most people seem to do this according to the 
PEG/LiAC/ssDNA method by Daniel Gietz. You will find a detailed protocol
on the Gietz lab trafo homepage at

www.umanitoba.ca/faculties/medicine/human_genetics/gietz/Trafo.html

I posted a newsgroup request concerning the same problem a few weeks ago
and got lots of response.
Most researchers made good experiences with the Gietz protocol though some
seem to have the same problems we have.
Critical steps seem to be:
-preparation of reagents, especially carrier DNA
-kind of bait vector used, pAS1 turns out to be not very stable in yeast so
efficiencies are rather low
-eposure of the agar plates to light lowers the efficiency dramaticly. That
is what Dan Gietz found out and he assured me that it is really very important
to keep the plates in the dark before use.

You can find a step by step outline of the use of the two-hybrid system in

Gietz et al., Identification of proteins......, Mol. and Cell. Biochemistry 172:
67-79, 1997

Hope this helps
Ricky 

******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiologie
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
e-mail: boernke at ipk-gatersleben.de
http://www.ipk-gatersleben.de





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