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

Frederik Boernke boernke at
Thu Jun 5 12:33:17 EST 1997

>Jeffrey David Bray <bray+ at> 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
>>> 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
>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.
>Joe Boutell.

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

More information about the Methods mailing list