kindly suggest some changes that can be done to get proper result and proper melting.
i am not able to get some shift in temperature for melting of different standards.
I am trying to standardize HRM for methylation since last few months but have not succeeded yet. the problem that i am facing is that there is hardly any difference in melting in 100% methylation standard compared to 0% methylation standard. even mixed standards with different methylation %ages and unknown patient samples also don't show any difference in melting. all these standards and unknown samples show melting around 76C.
I am using LightCycler480 from roche and their HRM dye for this technique.
the conditions that i am using are as under:
54C annealing temperature, 3mM MgCl2, 0.15 micromolar primer concentration.
Kindly suggest me some changes needed to be done to get the proper shift in melting temperature for different standard
your positive approach will be highly appreciated.