Siddharth is right the best way to optimize the annealing temperature is to do a gradient PCR. We usually use a range of 8 different temperatures with Tm-5 degrees as the middle temperature. We do normal PCRs to optimize out annealing temperatures. We run these on normal 1% agarose gels and when we have decided on the optimal temperature we then move on to the qPCRs.
Optimal annealing temperature is usually about 5 degrees lower than the lowest primer Tm, but further optimization may be needed.
Generally, an annealing temperature about 5°C below the lowest Tm of the pair of primers is used.but some time greater than primer(s) melting temperature. but further optimization may be needed.
For qPCR, the optimum annealing temperature of primer pair should be 10 degrees lower than the probe Tm if its a probe based chemistry and as said above, generally 5 degrees lower than the Tm of primer pair. Also ideally both the primers should have same Tm. But these values may vary and optimisation is almost always needed. best way is to first optimize your primer pairs on normal PCR before going for qPCR.
you can set up PCR reactions with lower dilutions of your template at 2 degree decrements and see what temp gives you the best sensitivity/ LOD and least background fluorescence
Siddharth is right the best way to optimize the annealing temperature is to do a gradient PCR. We usually use a range of 8 different temperatures with Tm-5 degrees as the middle temperature. We do normal PCRs to optimize out annealing temperatures. We run these on normal 1% agarose gels and when we have decided on the optimal temperature we then move on to the qPCRs.
The best way to find out the annealing temperature is gradient PCR in the range of +/- 5C of the Tm of your gene. The tempertaure at which you will get the sharp and intense band will be the best annealing temperature for your gene.
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