help on yeast gene knock-out - (Aug/16/2006 )

Hi there,

I'm trying to knock out a gene from yeast using LiAc transformation with PCR fragment from Longtine's plasmid. Recently I've had the problem that when I checked those transformants growing up by PCR, some times I saw two fragment showing up from one single colony, corresponding BOTH a wildtype and a mutant!! I don't understand how can a cell be wildtype and mutant at the same time? I used a lot of DNA for transformation, don't know whether that might be potential problems....

Thanks!

Best,

Wei

Is it possible you are using a diploid yeast strain? That would explain the result.

I've thought about this before. But our strain is usually HO gene knockout so I think it hardly has the chance to switch the mating type and mate with each other.