I am using the sketa assay (salmon testes) as a specimen processing control for my enterococcus assay, but I am getting really horrible percent recovery (<1- 5%). These are environmental samples, and so I know inhibition can be a problem. I tried doing dilutions (1:1 and 1:4) but that didn't help at all, and I'm afraid to do any larger dilutions because my target sequence is at fairly low concentrations to start with. And I'm also already using BSA at a final concentration of 1mg/ml. I'm not sure what else to do to trouble shoot.
I'm also not sure that calculations are correct... this is what I've done:
Add 10ul of 500pg/ul salmon dna to 490ul extraction buffer per sample (results in 10pg/ul salmon dna per sample). Then add 5ul dna to 20 PCR reaction mix (results in a final concentration of 2pg/ul salmon dna per reaction). Does that all sound correct? So, to get percent recovery, I've been dividing the qPCR result by 10, then multiplying by 100. I'm wondering if I should be dividing the qPCR result by 2 instead of 10, but when I do that, my positive controls become >100% recovery.
Any help would be much appreciated!!!