yeast two hybrid

Hi everyone,

I am performing the "yeast two hybrid screen". Up to now it worked
well and I have isolated at least 12 independent clones. But now I
have a problem in reconfirming interactions. I performed yeast mating
and, although positive controls worked, I had no growth on selective
medium that allows growth only for cells expressing interacting
proteins. One possible explanation is that something happened during
storage at - 80C of the yeast strain expressing the bait, may be
the plasmid has rearranged (although it confers the ability to grow on
selective medium that allows to select for cells carrying the plasmid
itself) or something like this. Is it a good idea to try again by
using a newly transformed strain? Or should I think to have isolated
false interactions (but I have succesfully restreaked them onto
selective medium and they had been collected only after 5 days from
transformation, thus decreasing the possibility of false
positives). There is anybody who faced with a similar problem? Any
suggestion will be useful.

Thanks,

Simona Baldassa (University of Milan)

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