2 replies to this topic

[Kenyon Applebee]

Hi there,

I am still getting a grip on all of the tricks and details of qPCR analysis.  I keep seeing a shoulder on the left side of my melting curves and I have not been able to figure out whether this is normal (melting of random cDNA secondary structure) or a sign of primer dimers.  I may have used too much cDNA in the run that produced the attached melting curve, but I would like to know if this could explain getting >100% efficiency (slopes of ~2.8 on log(cDNA dilution) vs Ct), or if I have primer dimers.  None of my no-template controls had any peaks in the melt curve, which is why I haven't just gone ahead and designed new primers (plus two of these primers I got from another lab's publication, suggesting they work)

The melt curve includes data from a plate with three different primer pairs.

Thank you!

-Kenyon

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[Trof]

Your melting curves look normal, the shoulder is always there. I don't see any dimers (but the lower temperatures are a bit obscured on the graph, to many curves). I don't get why you abandoned your first primers when you have no peaks in non-template control, there should be none.
Reason for >100% efficiency could be inhibition in higher concentrations, or I often see it in really short amplicons (like 60-70 bp). When it's not bigger than 110% you shouldn't worry about it IMHO.

[dtae]

To see whether the >100 E is due to standard curve artifacts you should consider calculating well-specific E values (e.g. with LinRegPCR if your software doesn't support it).

D