Yeast 2-Hybrid Screening

Frederik Boernke boernke at nospam.ipk-gatersleben.de
Wed Nov 11 21:17:23 EST 1998


Barbara Gorfajn wrote:
> 
> Ricky,
> 
> I have a few more questions, and some answers for your questions.....
> 
Hi Barbara,

unfortunately I have no experience with CG-1495 so I cannot say any
thing about the 3-AT thing. I am using Y190 which grows like hell if
not at least 25 mM 3-AT is added to the plates.#
Concering the lacZ-assay I rountinely plate His+ colonies onto x-gal
containing plates, works great with my strain. Positiv colonies usually
turn blue within a few hours (minutes) to overnight. I am afraid there's
not much hope if nothing happened after 24 h. But to be sure you should
do the filter lift stuff. I tried two different ways which both work
quiete well. One is the "classical" with transfering the colonies onto
a filter paper after 2-3 days of growth, the other is plating the
colonies directly onto nitrocellulose filters placed on the 
respective plates.
In my opinion is the x-gal plate assay much nicer, because you'll get
the results much quicker than by doing filter-lifts.
I also heard about the lesser sensivity but I never cared.

Hope this helps,
Ricky 

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

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



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