>100% qPCR efficiency
Posted 13 May 2011 - 06:00 PM
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.
Posted 14 May 2011 - 08:00 AM
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.
I never trust anything that can't be doubted.
Posted 17 May 2011 - 08:45 AM