Possibly someone can explain this to me...
I need to prepare some serial dilutions of standards for generation of standard curves, which will be later used to estimate abundace of 4 genes in complex environmental samples using qPCR. I have cloned these 4 genes into plasmids, purified and estimated concentration of the isolated plasmid DNA using a Nanodrop (spectroscopy) and Qubit (fluorometry). I normalised the concentration for each plasmid,prepared 10 log dilutions and ran qPCR with them. I have observed, however, that Ct values for standards containing the same estimated concentration of the template(e.g. 10E8 copies/ul) for each gene were significantly different. The difference was up to 6 cycles. The amplification efficiency was similar (1.90-1.94). Gel analysis of the plasmid DNA showed a single band (no signs of RNA or bacterial genomic DNA contamination). The same machine and PCR mix was used in all reactions.
Can anyone explain such great discrepancy?
Edited by skolyar, 21 November 2010 - 06:03 PM.