my FW and REV primers Tm are 53.69 and 60.98 respectively based on the manufacturer's sheet( of course the TM(s) are different in NEB or primer blast). Unfortunately, my thermocycler does not have temperature gradient. So I started a PCR with 58C without any band, in 57C I saw a weak band together with nonspecific bands. In 56 and 60 C , I did not observe any band. Would you please help me in this issue ?
Hi Faezeh, on the Cycler we implement annealing temperature Ta not Tm. The annealing temperature (unique for a primers pair) should be low enough to allow both forward and reverse primers to bind to the single-stranded DNA, but not so low as to enable the formation of undesired, non-specific duplexes or intramolecular hairpins. Thereby, the annealing temperature is usually set as a few degrees (3-6) lower than the lowest Tm of the primers.
You can get the Ta for the couple of your primers via online tools :
1-https://www.thermofisher.com/ma/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html
2-
https://tmcalculator.neb.com/#!/main
an essential of pcr is that both primers must anneal so the annealing temperature in your pcr must be below 53. If you cannot design a better primer then try starting at Ta of 50c and if that works then try higher annealing temperatures
The best way to find out the annealing temperature is gradient PCR in the range of +/- 5C of the Tm of your gene.
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