Yeast two hybrid X-gal Assay

[KarmaGirl242]

Hello all.

I am having problems with the X-gal assay of my yeast two hybrid. The assay has been done before by a former lab technician, and despite following her instructions, I am unable to repeat her results (which she was able to repeat).

 

I am re-doing the "confirmation" part of the yeast two hybrid protocol, in which I transform in my prey plasmid (from pACT2 library) into naive strain Y190. Next, I transform in one of several bait plasmids (vector pBG4D); these include vector only, and several constructs of our protein of interest (different domains, full length).

 

I am successfully able to transform in my constructs; they all grow on selective media (in my case, -his/-leu/-trp +3AT). After the second transformation (so now there is both bait and prey), I replate single colonies onto a fresh -his/-leu/-trp +3AT plate. From these plates, I perform an X-B-gal assay; placing nitrocellulose over the plates, dipping in liquid nitrogen, then placing on top of a filter soaked in Z buffer + X-B-gal. I leave the plates at 30[math]\circ[/math].

 

The next day, the only blue colonies are those containing the prey and the empty bait vector (my negative control is the only positive). Two days later, the filter is all blue on another negative, and partially blue on a positive control (the filter paper, not the colonies).

 

I've made fresh reagents, and am not sure where things could be going wrong, or how to troubleshoot this. The former technician was unsure of what to try, and she could not recall this problem in the past.

 

Any suggestions would be great, and thank you!!!

[Greippi]

Seems baffling from the info you've given, and I don't have much to suggest. You could perhaps try a completely new batch of plasmid (seems unlikely that would be the problem, but you've gotta troubleshoot everything I suppose).