Yeast-two-hybrid problems

[Adelaide_Science]

Hi All,

I am doing a yeast two hybrid assay with my gene of interest. But i have been having problems with characterising the interactors.

Using the minus 4 growth media i had colonies growing, and when transferred to x-gal they turned blue. All good so far!!!!!

I then went on to do DNA extractions and yeast plasmid isolations, and i have been unsuccessful in geting this to PCR up or transform into E coli.

I was wondering if anybody had had this happen to them or if anybody knows what else i should try to get to know what my interactor are (if they are actually true interactor at all)


Thanks

[Qiang]

Well, it seems to be pretty weard...

Did you use primers specific to the prey plasmid and do a direct PCR from your positive yeast diploid(mating)/cotransformant(cotransfo)?

Qiang