Two-hybrid: Many His+, but no lacZ+.

Frederik Boernke boernke at IPK-Gatersleben.de
Thu Jun 5 12:33:17 EST 1997


>Jeffrey David Bray <bray+ at andrew.cmu.edu> wrote:
>>Excerpts from netnews.bionet.molbio.methds-reagnts: 4-Jun-97 Two-hybrid:
>>Many His+, but .. by Frederik Boernke at IPK-Gat 
>>> I am just about to estsblish two-hybrid system in our lab. I screened 
>>> about 2 x 10e6 transformants and obtained about 120 His3 positiv
>>> colonies some of which show very robust growth behavior and apearred
>>> already after 3 days. I tested all these for lacZ expression in a filter
lift
>>> assay, but none of them turned blue. Is this something to worry about,
>>> or should I just go on to screen more?
>>> The problem is that I have absolutely no experience with this kind of
>>> stuff. Has anybody a suggestion?
>>
>>Well, I've doing some y2h and this is what I've determined:
>>
>>Screen more.  If they're not His+ blues, they're not what you want. 
>>Secondly you can suppress most the His+ whites by adding 5-50mM 3-amino
>>triazole to your plates.  The conc depends on your positive controls and
>>if you know how strong the interaction is.  I use 25 mM and still get
>>SNF1 and SNF4 interactions.
>>
>>Jeff Bray
>>Biological Sciences
>>Carnegie Mellon University
>>bray at andrew.cmu.edu
>>
>
>It may be helpful to know what strain of yeast you're using. I take it 
>you're probably using a Clontech system. If you've got their HF7c strain, 
>this doesn't go blue too well, but does give some large His+ colonies. 
>However strain Y190 is more sensitive in filter assay (around 5 times 
>faster when using the full GAL4 control plasmid) but requires a certain 
>amount (25mM) of 3-aminotriazole to cut out background His+ growth. So 
>you may be missing something because of the strain you're using.
>Cheers,
>Joe Boutell.
>
>
Hi!

I am using Stratagene's Hybri-ZAP kit which includes the SC strain 
YRG-2. The guys from Stratagene say there is no need to include 3AT
into the medium it would just inhibit growth, but I will try anyway. The
GAL4 control turns blue within about ten minutes, is this quick?

Thanks Ricky

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

Frederik Boernke
Research Group 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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