trembling dissociation curves in HRM assays - (Aug/12/2010 )

Hi evereyone,
We are starting developing HRM (high resolution melting) assays to quantify methylation in a given locus.
Primers were designed with appropriate softwares (MethylPrimer Express, Applied Biosystems) and assays were set according to standanrd guidelines (Wojdacz et al)with saturating concentrations of EvaGreen (Qiagen). The used thermocycler is a RotorGene 6000 (Qiagen). We obtain good amplification levels and after dissociation (65ºC to 95ºC ramp 0.2), apparently two differents peaks, corresponding to meth and unmeth products. Our problem is that melting curves appear not smooth with an ugly trembling. We tried to change primer concentration and annealing temperature, which slightly improved the quality of curves, but not significantly.
We will thank any advice or insight about how to fix our curves!

Rocio

Could you post a picture of your melting curves?

ElHo on Fri Aug 13 15:00:00 2010 said:

Thank you very much for your reply. We were able to figure out the reason of the strange look of our curves: inadvertently, working solution of primers were dissolved with ddWater instead of TE! And that made all the difference. Hope this can help other begginners! I will post soon a picture of the uggly curves.
Cheers
Rocio

That´s funny! I always set up my primer working solutions in water (only stock in TE) and never encountered such problems.