I'm new here but desperate! I am using a 3-primer PCR to genotype knockout animals, with forward primers for the wild type and mutant alleles and a common reverse primer. This has worked well for a few litters but I have suddenly encountered a problem. The mutant band in my hemizygous animals has completely disappeared (I always run a known sample for both homo strains and a hemi.) I ran separate 2 primer reactions for each combination and found that the mutant primer pair amplifies efficiently in the homozygous knockouts, but poorly in the hemizygotes - I do see a faint band but not enough to confidently interpret in unknown samples. This band cannot be seen in the 3-primer reaction. The wild type primers work equally well in homozygous wild types and in hemis.
If anyone has encountered this before, I would love to know why it's happening. I'm at a complete loss!
I extract my DNA from ear notches using the Qiagen kit.
All 3 primers have T m of 66-67 degrees and my annealing temp is 60 degrees.
I use Qiagen HotStar Taq and its standard buffer.
I have ruled out reagent and freeze-thaw issues by using completely fresh reagents.
Thanks in advance