QPCR Quantitation of copy number - (Aug/06/2007 )
I'm using SYBR Green on an icycler. I am trying to quantitate absolute copy number of an integrated virus in mouse genomic DNA pool. I get a good melt curve from both my sample DNA and a plasmid control. I've been calculating the plasmid copy number and then comparing that to the sample. I know how much total DNA I put in both reactions. Can I draw absolute copy number in this way from the infected mouse cells? I am also looking at mouse GAPDH for comparison, but I don't have a plasmid for that, just primers.
If the cells have ~6.6pg total DNA per cell (I read that somewhere), how do I do this calculation?
That sounds OK. I did quantify DNA concentration of biological samples by external standards but your procedure should work. Just get sure that the inserted DNA sample really contains this DNA concentration. You do this by spectrophotometry? A manual how to calculate the mass of a single haploid genome can be found at http://www.appliedbiosystems.com/support/t...f/quant_pcr.pdf . The other way should also work, but I'm not familiar with multiplex real time PCRs. An alternative are two PCR reactions with the two quantification systems (virus and GAPDH) and two separate standard curves in the same PCR run to finally calculate the ratio. You just make another standard curve from a mouse genomic DNA standard by serial dilution for the GAPDH gene and don't need a plasmid.