In gradient PCR i get amplicons at all temperatures from 50-60 degrees. Is this correct? Why this happens so? Which temperature should i finally fix as annealing temperature for future works?
Hello, dear. If you are looking for specific amplicons, the best annealing temperature is that generates a single band, with greater intensity, showing the expected size. Typically, specific fragments are amplified at higher temperatures. You could also test temperatures above 60 degrees.
Thank you Magnolia. I am getting a single specific band with the same intensity at all temperatures. That is wat the prob. So my question is which annealing temp should i use for future PCR reactions with this primer.?
Dear Vikashini Balasubramanian
You can also test temperatures above 60 degrees and between 30 to
65 degrees.
To me it appears that you have a very robust system, so there should be no problem. As proposed by others, you could try higher temperatures thereby shortening the ramping time and consequently the overall PCR time. If time is no issue just go with e.g. 55-57 C and you are in a safe range.
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