I ran HRM by using a primer pair with product size of 237bp. I used 15ng of good quality genomic DNA as template, but there is no amplification happening.
I should mention that this amount of DNA was previously detectable for my HRM when using a different primer with amplicon size of 100bp.
What could be the possible reason that there is no amplification detected in my experiment?
Can it be because of my primer and product size or sequence? My HRM kit, protocol, and real time pcr instrument are same as my previous successful HRM.
Try running a gradient end point pcr to see what temperature the primers really anneal at. They may be sitting over a polymorphism or just are a badly matched sequence. Possibly also the amoplimer may be high GC and need betaine or DMSO to help the reaction. Once yoy=u have a working pcr transfer back to the HRM
Paul Rutland, Thank you very much for your answer.
I actually did conventional PCR prior to HRM. The annealing temperature of primer pair was optimised at 55 degree C and I could see single band on gel for the amplicon size.
Was the working pcr run with exactly the same reagents as the HRM and also on the same pcr machine Nahid Tavana . Are any other researchers successfully working with the HRM at the moment. How long roughly have the primers been diluted for and are they in water or TE?
Paul Rutland,
The reagents and machine that I used for PCR was different from the one I used for HRM.
I am using LC480 (Roche) for HRM. I actually had a successful HRM (with another primer) at the same run as the unsuccessful HRM.
My new primer, from which I can not get results, is diluted in nuclease free water for about a week. However, my previous primer, which still work for me, were diluted a few month ago.
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