I'm sorry if the answer to this is something obvious that I'm missing - I am quite new to this and have been trying to figure this out for 2 weeks now.
I am using a standard taqman reaction to amplify viral genomes from dna isolated from mouse tissues (Qiagen DNeasy kit), and am getting very nice and consistent data within experimental groups, standardized to histone control. I am, however, getting very different calculated copies depending on which set of standards I am comparing to. I am amplifying a specific viral gene that has one copy/genome. One set of standards consists of this gene inserted into a small plasmid (2.5kb). The other standard is a Bacterial artificial chromosome (BAC) and contains the entire viral genome (>100kb). The purity of my standard DNA seems OK. However, I get very different results from these standards (1.5-2 logs difference) with the smaller plasmid giving me higher genome copy reads in my samples. The dilutions and calculated copies have been checked and rechecked. The standards both give fantastic curves, but are very far apart. The most obvious answer is that they aren't at the concentration I think they are, or aren't diluted properly, but I have checked and rechecked this. What gives?