SSRs has different melting temperatures of forward and reverse primers am not getting results when i put average Tm as given for both.
If you have access to gradient PCR, use that to find out the exact Ta. If not, then try setting up different PCR reactions with varied Ta.
I think that this is possible only through empirical checking
Hello,
I could recommend two options:
1. gradient PCR
or
2. to apply PCR program divided into two parts, first in which you should apply decreasing Ta (e.g. 15 cycles with decreasing Ta from 65 to 58* with -0.5°C per cycle) followed by ca. 20 cycles with constant Ta = 58°C*).
* varied Ta estimaded based on melting temperature of used primers or gradient PCR (sometimes this "combined" program impoves the PCR efficiency).
Good luck
Piotr
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