I'm doing homologous recombination in yeast and have got a rather strange result, and I was hoping someone could shed some light on it. To screen my yeast for positive recombination, I use PCR primers flanking the recombination site and should get a 2kb band on my gel if there is no recombination, and a 1kb band if recombination has occurred. The 1kb band encodes phleomycin resistance gene.
Weirdly though, I am getting double bands in a lot of my colony PCR screens. I get both the 2kb and the 1kb band. I even took one of the colonies that did this and streaked it out, screened those colonies, and got the same problem again.
Has anyone ever encountered this, or does anyone know what's going on?