I am using Clontech's One Hybrid Kit to clone two transcription factors. I have generated a dual his3/lacZ reporter strain and just done a library transformation with my dual reporter strain.
Despite finding that 45 mM 3-AT inhibited background his3 expression in my dual reporter, I obtained a thick pink lawn on all my library selection (his-/leu-/45mM 3-AT) plates, with some larger white colonies on top of the lawn on some plates. I've restreaked these white colonies and done a B- gal colony filter lift assay on these colonies but none of them went blue!
Has anyone had this problem before or any suggestions why these white colonies grew so large on plates with 45mM 3-AT??!
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Yeast One Hybrid
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