I used four primers (Primer 2,4, 5 and 6) with 2 rice variety and tested its annealing temperature at 53, 54, 55, 56,57 and 58 degree centigrade on PCR. The photograph of the gel electrophoresis is attached here. First four primer in the figure is rice variety 1 and later four primer is rice variety 2. So, please suggest me the best annealing temperature of the primer 2,4,5 and 6 best on the attached file. This experiment is done to identify blast diseases resistant gene on rice and the primers are SSR marker for blast resistant gene of rice.
the best result looks like 53c but even at 53 two markers do not work on sample B so I would try 51, 49 and 47 to try to find an annealing temperature that amplifies many strains of rice hot just strain 1. Your dna is good as shown by some amplification in primer 1 and 2 so use sample A as a control and test lower temperatures for the not working primers on sample B
They have a fairly wide optimal annealing range of 56-58 °C. For the second rice variety, either check the quality of the DNA (because there is no PCR product), or for this variety the PCR product does not form at all (maybe these are primers for qualitative signs ???)
Are the different primers addressing the same gene?
If yes:
Primer 4 seems to give specific products for both varieties at 53°C. It fails to give any product for variety 2 at higher temperatures. So I think you can use primer 4 at 53°C for both varieties.
If no:
As Paul said, you might test even lower annealing temperatures. However, I think that the primer sequences don't fit to variety 2. You should possibly design new primers specifically for this variety.
Hi Ajaya. I agree with Farhan. I think, carrying out gradient PCR experiment would be the best thing to do
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