I am trying to amplify my target gene from viral DNA for making a standard curve. I have used qPCR (Sybr system) instead of ordinary PCR to monitor the specificity. Regarding the dissocation curve, the peak corresponds to genomic DNA template has a melting temperature at ~81.5 oC.
After that, I have purified the PCR products using gel purification kit. A 10-fold serial dilution of the amplicon have been done and they are subjected to a second qPCR (with the same reagent and thermo-cycling profile as the previous one). Surprisingly, everything goes well execpt the peak at the dissociation curve has shifted to ~85 oC. Is it contamination? Or the melting temperature of my target gene has changed after purification? Attached please see the dissociation curve for that. (I'm sorry for the low resolution)
I have identified the amplicon by RFLP and it is the gene of my target.
Comments are always welcomed!