I am new in this forum hoping to find some answers.
I just started a Yeast 2 Hybrid Protocol using the Yeast strains Y187 and AH109 and the Plasmid pGBKT7(Gal4-BD) with my gene of interest.
One of the pre tests is the X-gal test. I have used the Protocol-at-a-Glance with alpha-X-Gal and both strains turned blue if they contained the Plasmid (empty strains were still white).
Now I am confused because I thought they have to be white as long there is a interaction between the Bait and the Prey Plasmid otherwise its auto activation. This would be worse because it means I couldn't do the Y2H-screening and I am running out of time.
I am also wondering why the colonies containing sequence A in the Bait Plasmid are much more blue than colonies with sequence B.
Does anyone has experiences with that? I just read that Promoter stuff again (Mel1 ...) but I am still lost.
Thank you all!