I am trying to optimize a SYBR assay and one primer set is giving me dimers every time. I've increased the annealing temperature to close to the calculated value for that primer set, and the other set is running great so I don't want to mess with the thermal profile any more. I've already decreased the primer concentration to 50 nM (25 ul reaction) and there are still dimers amplifying around Ct 30-35. I know the next step is back to primer design, but we are hoping to compare this work with another published paper that used the same primers. Maybe I just need someone to tell me the hard truth that it's time to give up and redesign. Thanks!
Hi!
Optimizing the concentration of primers allows for improved efficiency of the reaction; if the set of primers generates dimers, it's best to redesign it.
I agree with Brandon Morales-Merida : redesign your primers.
My approach is typically to design two or three pairs to my target of interest, and try them all out, with the aim of finding at least one pair that works under my standard PCR conditions. I will then use that pair.
For the rest, throw them away: you've only wasted ~£5-10 per pair, and you've saved yourself a lot of time and reagents you would otherwise waste trying to optimise conditions for a fussy pair of primers.
Thanks Brandon Morales-Merida and John Hildyard! Time to swallow the bitter pill. I did find that we are only getting dimers in wells with no template. Sample wells and NTC wells have different distinct peaks on the melt curve. I'll still recommend redesigning them, but I'm curious if that changes your thoughts.
I really like the idea of starting with multiple primer sets though. If that saves even a few wells of master mix it's worth the cost (not to mention time).
I suppose it really depends on _when_ you are getting primer dimers.
Given that PCR is an exponential process once there's even a single template there, if you run a reaction for enough cycles, you'll almost inevitably amplify something.
I run my qPCRs for 40 cycles, and I will occasionally see signals (and melt peaks) consistent with primer dimers in my negative controls, but these usually only emerge after 36+ cycles. Given that my actual positive signals emerge at much earlier cycles (20-25), I can assume that even if primer dimers are contributing to the fluorescence in my actual samples, those primer dimers are contributing very, very late in the process, long after the 'quantitative' bit of the qPCR has been determined.
I mean, the ideal world is 'no primer dimers', but still. If you see primer dimers emerging in the late 20s/early 30s, it's perhaps cause for concern, but anything after cycle 35 is readily identifiable as non-specific amplification.
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