[Arabidopsis] forum question about yeast-one-hybrid

[Inge De Clercq]

subject: _yeast-one-hybrid beta-galactosidase assay

_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