Mutagenesis, B-gal, & 2-Hybrid Questions

MIke mike at ncsu.edu
Tue Feb 18 15:05:04 EST 1997


Hello,

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.  

Specifically, how do I get rid of all the dead/nontransformed cells
before plating?  

***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?


***Another interesting thing that the guy was doing was PCR
mutagenizing the components of a known positive interaction and then
transforming the PCR product and the linearized vector into the yeast,
where they spontaneously/naturally recombine.  Anybody else ever done
this.  Seems like a great way to investigate specific interactions
between your two proteins.  


Thanks, 
Mike Faughn
Dept. Genetics, North Carolina State University
mike at ncsu.edu




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