Melt Curve Analysis - (Feb/21/2006 )
I have recently started Real-Time PCR using SYBR Green I. I get a peak in my melt curve and a smaller peak at a lower temperature (more like a shoulder ). I ran the products on a gel and I got an amplicon of the correct product size. Should I worry about the extra peak? Would this be publishable?
if you could attach a picture it would be helpful?
would you expect the secondary peak to be primer-dimer?
one thing to note is that secondary peaks are pretty much always bad juju unless they are REALLY tiny; this secondary product or primer-dimer will be read by the machine as extra target...resulting in lower Ct...resulting in skewed data
this is the main problem when working with SYBR, you have to get really really tight specificity
If the GC content of your PCR product varies significantly over the length of the fragment, then the Tm will show multiple peaks or a wide hump. The effective range of the GC averaging is about 50 bp or so.
Plot the isochore of your sequence to see if this is an issue.