I am developing a qPCR assay intended to detect virus templates in mixed samples. I have created an oligonucleotide standard (single stranded) for the assay, and quantitated it using the RiboGreen Quant-it kit. I have been performing test runs of a standard curve of my olgio and comparing it to genomic DNA that was quantitated using the dsDNA pico green Quant-it kit. I beleive that I am putting the same number of copies into the genomic reactions as I am putting into the oligo reactions, but the oligo is consistently running one log worse than the genomic! For example, if the Ct of 500,000 copies of my genomic is 21, the Ct of 500,000 copies of my oligo is 24.3. I expected equal copy numbers to produce very similar Cts.
Has anyone seen this behavior where genomic acts differently than oligo before? Any suggestions on what to try?
I did the math to figure out copies per reaction based on the molecular weight of each template type, quantitated both templates in-house before use, and checked that the efficiencies were "similar". What else can I look at?
Edited by katurne2, 11 June 2012 - 12:52 PM.