I have designed a pair of DNA primers to amplify 700bp region in a fungus. When I tested these primers they produce heterogeneous amplifications among different strains, in other words amplify great in one fungal strain and not so much in another. I checked the quality of DNA of samples I am using, everything is fine. I realize that one of the possibilities could be the fact that there is a lot of polymorphism in this region among different strains. But the region I am trying to amplify is a protein coding DNA. That tells me that there should not be a lot of polymorphism that causes PCR failure among fungal strains of the same species. I also tried to add MgCl2 to my PCR reactions. Regardless of the MgCl2 concentration (0.5, 1, 1.5, and 2 mM) PCR completely fails when the reagent is present in the reaction. This does not make biological sense to me, since I would rather expect non-specific amplification with MgCl2. Unless it causes primers to bind to different parts of the genome? I will be very grateful for any input and thoughts about what may be going on and where should I move next. Design a different pair of the primers? Or is there anything I am missing to fix the problem? Thanks a lot!