I am trying to standardize the annealing temperature of a Primer. In 53 degree,a clear band on has been found at proper position (500 bp to 600 bp) but a non specific band appeared within 300 to 200bp. I have run Gradient PCR (52,52.8,53,53.2) but in all temperature (except 53 degree) gives a smear product with primer dimer.
The primer pair are
Foreword-GACTGTGACAAAATCCCNTTCCA
Reverse-GGTCTTCATCTYHGGYTTACAAGAC
Please help to solve this problem
Thanks in advance
As per the primer sequences, the calculated annealing temperature for your primer is 63 degree celcius. You can try one more round of gradient pcr for temp 62, 64 and 66. hope this woild work for you.
You can make use of this website https://tmcalculator.neb.com/#!/main to calculate Tm for primer set.
Rahul Bodkhe i have also run the PCR with 60 and 61 degree celcius but result showing no band. I think above this it will not give any PCR product.
95°C - 5 mins
95°C - 30 secs
60°C - 50 secs
72°C - 40 secs
72°C - 7 mins
Did you tried this?
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