The set of primers are well designed with the sequence and even after doing Primer Blast , they are showing only one specific amplification .
Set up PCR reactions with different annealing temperature or else do step down reaction
In my opinion, PCR reactions gave non specific products due to lower annealing temperature. you should calculate the annealing temperature manually by counting the individual bases of each primer and use the lowest temperature of the pair. otherwise if you have a gradient PCR machine then you should use a gradient PCR (65-56 C annealing temperature) program to check the best annealing temperature for the primer pair. Hopping it will help you to figure out problem. best of luck
Thanks Manjula and Haider for your valuable suggestion.
I did a gradient PCR . It clicked well but still very faint amplification signals are seen. I am trying to lessen down the no.of cycles and primer concentration to omit out the above mentioned result.
One more option is there, you can extract the specific band from gel and use that as template. reducing the number of cycle is not a good idea if your using the product for cloning.
Try hot start PCR: add the enzyme during the extension step. This will prevent primers that bind unspecificaly to be elongated at room temp or during the annealing step (the polymerase has residual actvity at temps lower than 72 deg C). The hope is that only perfect matches will survive the 72 deg C and those will be elongated preferentially. It might not solve the problem completely, but it may improve the amplification of your product. try with a small 50 uL reaction and see how it works. Good luck
Well I had reduced the primer concentration. It clicked well. Thanks all once again.
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