Blog post — "Fixing our genotyping PCR failures" (labnotes.example.com) For the last month our genotyping PCR for a floxed allele has been giving us faint or completely absent bands, even on samples we know are positive from earlier litters. We traced it back to two issues.

First, our ear-punch/tail-snip lysis buffer had been sitting at room temperature for weeks instead of being stored at 4°C, and the proteinase K in it had likely lost activity — fresh aliquots restored strong, clean bands immediately. Second, we were eluting DNA in too large a volume for old or tiny ear punches, which diluted the template below a usable concentration; cutting the elution volume roughly in half for small samples brought Ct values back into a reasonable range on downstream qPCR runs.