yeast two hybrid

Alexandra Blak blak at gsf.de
Wed Aug 27 01:15:00 EST 2003


Dear yeasters,

after trying a YTH screen with a pretransformed library, that didn`t
work so well, I created my own cDNA library by using Clontech`s MM
library construction and screening kit and I have to say that the
screen worked really nice (after having a lot of troeble with every
single step)!  But now my problem is: I got around 500 colonies and
although I theoretically know what to do now, like PCR, extracting the
bands out of a gel, cutting them with Alu or Hae; colony hybridisation
(with what?), I am really worried about that number - 500. Does anyone
of you know a better and faster way of sorting out false positives? I
restreaked my colonies and most of them still grow. alpha-Gal assay
didn`t work on plates - they are all getting blue and for beta Gal
assay I made the same experience. Did somebody also use the MM library
construction kit?  I would be very very thankful for every little
hint.  Thanks a lot in advance.  Alexandra

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