This should help..

I used genotyping 5 SNPs by real Time PCR HRM using Evagreen and Qiagene Kits.It could help you: Mutation Scanning by High Resolution Melt Analysis: Evaluation of Rotor-Gene 6000 (Corbett Life Science), HR-1 and 384-well LightScanner (Idaho Technology)

We always used SYTO9 but I'm just marking a thesis that is using EVAgreen and I see others have posted protocols. This may also help:

www.gene-quantification.de/hrm-dyes.html

LC-Green is alternative.

Hi, i used evagreen from metabion and it works fine

I used the SensiMix kit from Bioline that includes EvaGreen and its very good for A-T and A-C SNPs

I've genotyped a number of SNPs using HRM. In my experience, the primers and instruments are your two biggest factors for generating a successful genotyping run. The smaller the amplicon, the bigger the temperature shift in general, so good primer design is key. For dyes, I typically use Promega's GoTaq qPCR Master Mix (which includes BRYT Green). In my hands, it gave the same results as Eva Green, and had much brighter signal than SYBR. Plus it's formulated to be resistant to inhibitors and thus is more tolerant of poor DNA preps, or sample-to-sample variation in preps.

Otherwise, the protocol and optimization you use is almost identical to regular qPCR. Just follow the instrument directions for setting up the melt curve appropriate for HRM.

p.s. Many instruments specify that you have to use THEIR qPCR mix, but we've used Promega's BRYT Green (in GoTaq qPCR MM) and EvaGreen on BioRad, Corbett, and Roche instruments with no issues.

Thanks john

the link that you provided (www.gene-quantification.de/hrm-dyes.html) is very helpful.

Thank you all, for your comments!!!!!!

We are using the KAPA HRM High Resolution Melt mix from KAPA Biosystems, which works extremely well in our hands and is rather affordable. As was emphasized by others, short products under 100 bp work best.

If you used protocol for a 20μl reaction, Prepare the reaction mix by combining 10µl EVA Green Master Mix, 0.8µl primer at 200nM final concentration (For and Rev) and add the remaining 6.4 µl Nuclease-Free Water. Carefully pipette 18μl of reaction mix into each reaction well and add 2ul of your template. Then program the thermal cycler with the desired thermal cycling program  before you put the plate in thermal cycler to start the amplification. Then When the run is complete, analyze the data using melting curve analysis.