Dear Bhargav, I usually thawing of freezing solution: firstly keep on ice-water a few minute, then keep it warm by our hand and gentle, even slowly but keep the solution not shock it as proper.

Anyway, before I keep the master mix or other solution on freeze  -20oC into - 80oC, I always divided it   into small volume at 5-10 micro-tubes, just for safety keeping the solution thawing-freezing many time.

I never have trouble thawing-freezing way, hope you get a good result soon

regard

fatchiyah

Brawijaya University

Is the only variable you are changing the master mix? (For ex, you aren't making new primer substocks each time -- you are using the same substock with each mastermix)? This will rule out other factors.

Typically, qPCR mastermixes are not to be re-frozen after the first thaw. Check your manufacturer's guidelines. You can usually put them at 4C for the next 2-4 weeks. However, some manufacturers do test the number of freeze thaw cycles before any significant loss in performance, so also check that. The major issue with freeze/thawing is the stability of the dNTPs. The pH can change with temperature, depending on the salts used, and hydrolysis more readily occurs once thawed. If these come as a separate vial, then DEFINITELY make single use, 10mM aliquots after the first thaw. This is good molecular biology practice. However, most manufacturers do have improved formulations to prevent degradation, so I find it hard to believe that each of three mastermixes tested would only work after one thaw. It seems like there may be another variable unaccounted for here.

Thank you Fatchiyah and Jo Lynne Harenza for the answer. 

First of all, I accept that I did not made aliquots. Next time I will do it. I have changed not only master mix but also primer stock. Even I tried it for different primers also e.g 357F-518R of 16S rRNA and another two pair of primers for mitochondrial HV-I and HV-II regions also but not result in any of the primers with any of the master mix.  

Good molecular Biology practice is essential. preparing more aliquots is very useful. Dont freeze-thaw your stock many times.

Perform some gradient PCR to check if the annealing problems.

Thanks

Thanks!

I have tried gradient more than 10 times ranging from 47 to 65 °C.