Hi everybody
I'm trying to genotype two SNPs with HRM. one of them(SNP1) is set up but the other one(SNP2) shows different melt curves in DIFFERENT temperature(some samples in 92 C ,some in 86 C and some don't amplify at all) although it was set up 3 months ago and melt curve was repeatable in 92 . it is surprising that with different melt curve I see same band in gel . I use same conditions, DNA concentrations, master mix...
Depending on how your oligos were produced and cleaned-up, you could have an n+1 component in one of your mixes. In other words, one of your primers isn't pure and contains oligo fragments that have one extra base attached.
Hi Pardis
Looking at the figures, and considering that primers were tested/validated on known samples (as you wrote) there may be something going wrong with fluorescence reading, probably related to DNA of your samples. Even the right melting curve shows indeed a "glittered" baseline. The fluorescence peak is very weak. Is the DNA coming from environmental samples? Did you include in the plate some positive internal amplification controls and non-target controls in order to compare the results? Primer dimer/concatamer can also affect the reaction, as well as presence of extra-bases as Randolph rightly wrote. Use primers at least HPLC-purified, use some internal controls (some gene specific to your organism) and negative controls (water can be ok), check the quality of samples DNA before going to HRM.
Lastly, if you have bands on gel, try to sequence them to understand if primers rightly amplifiy the specific region (and not an aspecific DNA). Hope this help, good luck!
PS Remember to use specific reagents for HRM. Sybr Green is not always the best choice, Eva Green is better. And also white plate are better than clear plates for HRM analysis.
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