yeast one hybrid - beta-gal assay

[inge]

Dear all,
I am performing a yeast one hybrid screen according the protocol of Deplancke with histidin and beta-galactosidase selection. After screening with a cDNA library, I picked up some colonies on medium lacking histidin + a concentration of 3-AT at which the strain does not self-activate. I retested these positives on a new plate with the same composition: a few colonies could not grow (which seems to me a negative control that is ok), but others again growed well, even a higher concentration of 3-AT... So, it seems to me that there s nothing wrong with this first test.

The problem is that all these colonies don't give blue color after performing the beta-gal assay (protocol according to deplancke, 2006 and clontech: colony-lift filter assay). I tried already multiple times.

Does someone have an idea what could be wrong? The construct containing the LacZ gene is ok: confirmed with PCR, and some strains were blue during testing the self activation in the beginning. Could it be that maybe my not-self activating strain (that i selected for performing the screen) is contaminated with neighboring colonies at my master plate that were self activating, and that the colonies that i picked up after my screen are just self activating colonies instead of positive transformants?

Many thanks in advance...
greetings,
Inge