Y2H beta-gal is blue but when retransformed is no longer blue - Yeast-two hybrid troubleshooting (May/02/2008 )

Hi. I did a Y2H screen to find novel interactors for my protein of interest and I have run into a problem.

The procedure I used was as follows:

1) Selected colonies that are positive for growth on -HIS and blue in the beta-gal assay.
2) Isolated plasmids from those yeast colonies.
3) Transformed that plasmid mixture into E. coli, performed colony PCR and selected the colonies that contained my prey (cloned into pGAD424)
4) Bacterial mini-prep to isolate my plasmids
5) Re-transformed the putative interacting plasmid into yeast that contained my bait.

When I do the beta-gal and the growth on -HIS..they are not blue and they do not grow well on -HIS.

Where did my putative interactor go?

Any suggestions on how to get this putative interactor back? I suspect that when I isolated plasmids from yeast it was lost...but I can't figure out why.

Thanks to everyone!

It's possible that your original hit had more than one pGAD plasmid, and you isolated the wrong one from bacteria. But, I really hate the 2hyb system. Have wasted too much time with it, and will never touch it again. It is great in theory, but in practice it sucks. If you are doing your PhD or a postdoc, I'd suggest finding a different approach soon.
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