Mutagenesis, B-gal, & 2-Hybrid Questions

Martin Gossen martin.gossen at rz.ruhr-uni-bochum.de
Wed Feb 19 16:32:02 EST 1997


On 19 Feb 1997 07:48:12 -0800, Mike at UNITY.NCSU.EDU ("Mike") wrote:

>I am conducting a 2-hybrid screen using strain PJ69.4a.  When I do a
>large scale transformation with decent efficiency I end up with a lot
>of cells.  I usually spin them down out of the PEG after the heat
>shock and resuspend in water, then plate them out.  The problem is
>that I am putting so many cells on each plate that they "glop up", so
>much so that it might impair the growth of positives or obscure their
>detection.  If I dilute the cells and use more plates then I will have
>to use *a lot* of plates, more than seems reasonable.  I am
>considering taking my transformation and letting it recover in drop
>out broth before plating, this won't cut down on the number of cells,
>but may enrich for positives.  I know a drawback here is that strong
>positives will be enriched for disproportionately, meaning that I
>would have to screen more of the positives to find out if there were
>multiple proteins interacting with the bait.  But right now anything
>at all would be welcome.  Any feed back on this situation would be
>welcome.  

Sounds like your strain (I never heard of it) does not effectively
select against Histidine deficiency. Try a strain that selects well
against cells without an interaction. I recommend YRG-2 from
Stratagene, though you will encounter other drawbacks here, namely the
lack of an effective beta-Gal test (not very strong signals and not
always reproducible).

I think your strategy propsed will not work, as your strain does not
select against cells without an interaction, so all transformants will
grow in dropout.

>***Also, I recently saw a seminar that had slides of blue yeast
>growing(?) on media.  Was the X-gal included in the media.  I thought
>that B-gal wasn't excreted by yeast and that they had to be lysed,
>what was going on here?

It does work without lysis but the signals are much weaker (that's
what Clontech say).

Martin Gossen
Molecular Human Genetics
Ruhr-University Bochum
Germany




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