PCR DNA Concentration - (Apr/11/2013 )
I have a set of mtDNA Taqman assays for various animal species and I'm trying to validate them. Because this assay is qualitative, I have not been diluting down the DNA after extraction (from muscle tissue) before PCR. The assay has a good sensitivity (~0.05% target in mixed species tissue). However, some of the assays are giving non-specific amplification at around Ct 30-35.
Could this non-specific amplification be due to running too high concentrations of non-specific DNA?
If I dilute my DNA (to 10ng per rxn for example), am I likely to lose my 0.05% LOD?
Unfortunately, I can not help you. But I would like to ask you about protocol (or citation) or recommendation for Taqman assays for quantification of mtDNA in human muscle samples. I am just starting this method and any advise would be very helpful for me. Thank you.