So I am genotyping a standard gene targeting KO and am having issues. Sometimes the PCR reactions work perfectly even as multiplex reactions and at other times I get non-mendilian proportions. The LacZ band is almost always nice and bright, however the WT allele I have trouble with, in a breeding of -/- and +/- I get all hets for many mice and no WT and KO mice, though band intensity differs. I am not loading the same amount of DNA in all which I will correct however I figure that for the WT allele if its not there at all should show no band whatsoever. If anyone has any advice or has had this experience before please let me know! Im thinking I should redesign primers but what is baffling is that it used to work and still does on occasion, but mostly it does not.