In article <9603011353.AA20358 at kaa.crbm.cnrs-mop.fr>, fesquet at KAA.CRBM.CNRS-MOP.FR (Didier Fesquet) writes:
>> during a two hubrid screen, upon recovery and Ecoli transformation of
> plasmid from his+lacwhen we transform back yeast to check for specific interaction. the plasmid display still HIS+ phenotype but not lac+.
> does anybody encounter this problem. how can we explain this?
I also observed similar problems.
After retransformation of a certain library plasmid into the screening
strain, which already contained the bait plasmid, everything was okay.
After retransformation of BOTH plasmids into the starting yeast
strain, all of the transformants are able to grow on -His+3AT, but
some of them are deep blue, some are blue, some not blue at all.
I think this phenomenon is due to the number of the 2 mikron based
plasmids which is variable in the cell.
I wouldn't worry and go on for secondary assays if one of your true
positives looks like a strong activator in most cases.
Institut fuer Biochemie I