I'm trying to determine copy number of virally infected DNA in cells that I have isolated DNA from.
For my standards, I have 2 templates. The first is a plasmid carrying the viral gene of interest. The plasmid is 10kb long, the DNA sequence being amplified is 176bp.
The second standard is the 176bp amplicon itself, which I amplified, ran on a gel and cut out.
I speced both the plasmid and amplicon DNAs and calculated the copies per pg of starting material.
When I run QPCR on the same 'copy number' of each template, I get different crossing points. I get nice standard curves for each, but they are off from each other by orders of magnitude!
I can't figure out how many copies of the gene I have in my test samples if my 2 standards are so different!