I am having a lot of issues with genotyping a particular mouse line in our lab. We obtained the mice from Jackson labs, and have always used their genotyping assay and primers, which have been fine for the past 4 years. Recently I have been trying to collect mutant embryos by breeding 2 heterozygous mice together, and was not able to get any mutants according to PCR genotyping. So, I went back and had a company regenotype all of my breeders to make sue they were hets. Every single one came back as a WT, meaning that I have lost the line, and it is no longer available for purchase. We are stopping the experiments, but I need to know where things went wrong when I defend my thesis next month.
We think that the problem was in the PCR design. It is a multiplex PCR, with 3 primers, that gives a WT and a mutant band which are only about 100 bp apart. Hets will have both bands. So when I see mice that genotype having both bands, I kept them thinking they were hets, which they apparently were not. When I ran the original genotyping for these, there are 2 bands. But when the company does it there is only a WT. Are there other things that the second band could have been when I ran the genotyping (shadow or stutter)?
Thanks for any help!