In my HRM analyzes, although I use the same samples in the same analysis, I get different Tm values. And I've repeated it many times, but there has always been a discrepancy. For example, even when I use primers with at least 40 bp polymorphisms, different genotypes can have the same Tm value. Can anybody give me a recommendation that I can get a normal Tm value? Thank you in advance.
Can you add more informations about your experiment? so we can help you.
Actually i had writen in explanation. If i repeat it. I have used real time pcr machine for doing hrm analysis. My sample is diploid wheat and i have some tailed ssr primer. For instance, when i used the population parents as reccurency at least three time times for optimisation of pcr, i am taking different meltng curve temperature value for each repeat. Normally i had to get same Tm value but as i sad this is the situation. And i could get different Tm valu from the its RIL in spite of they have same allels. I hope it could be more informative to explain mu problem. Thanks in advance.
So, actually did not use any negative control and now I am kind of a beginner and if you give me more information about negative control which is meaning and how can I use it in my work I would be appreciated to you. because I couldn't get any help from anybody and I trying to learn by myself.
Sure!
The negative control is the same PCR reaction that you used, lacking the DNA. If you get results from the negative control means that you get DNA contaminations so you can answer your question. Does it make sense?
1. Yes I mean not add DNA to the PCR mix.
2. Your reagents are contaminated with other DNA. Your results are not reliables.
I got. But it is impossible (contamination). Because I used different reliable DNA samples and still got discrepant Tm value. I think this method is so sensitive and its optimization is a little bit difficult. What do you think?
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