Protocol Online logo
Top : Forum Archives: : Real-Time PCR

dissociation curve analysis - (Mar/03/2006 )

unsure.gif Hi

I have a problem with the dissociation curve analysis. I run in the same plate cDNA and gDNA and I get the same pick in dissociation curve which means the same Tm for both cDNA and genomic DNA.
This it means cDNA contamination with gDNA?
The primers was selected in the same exon.


I analyse the g DNA a a reference matrix to determine the absolute number of specific cDNA molecules present in the sample. I use this gDNA as a standard.
It is a problem if in dissociation curve analysis a have the same peack for cDNA and gDNA?


if your primers were within the same exon, then you would expect your two products to be identical, therefore they would both have the same melting curve...if you want to absolutely rule out gDNA contamination in your samples, you need to a) design intron-spanning primers and cool.gif run appropriate controls

do you see what I mean? if the products will be identical, there's no way to know if there is gDNA contamination or not