I come to you beggning for a solution to this problem.
I am doing PCR routinely on a mutant Hprt locus in mice. The mutation is a deletion. We have a construct that can recombined to restore function to the mouse hprt locus and we need to know whether the deletion is there or not.
My problem is this: I am getting non-specific banding quite frequently with this procedure. The bands are pretty consistent between repetitions and one band is only slightly smaller than the band I am searching for, making it difficult to call a positive a positive.
I have essentially been told that without solving this non-specific banding, I will be seen as a weak PCR technician and fired, despite having no trouble with other assays.
I beg of you, forum team, to help me figure out what is wrong and what the best course of action is.
The only thing I can't do is to do hot start. While I am sure this would solve the problem, I am not allowed to purchase the more costly taq to solve this problem. Other members of my lab do not have this issue with the assay, even when they do it at room temp except for when they put the master mixes on ice prior to adding taq and plateing them.