We are getting same single band for multiple primers at a wide range of temperatures (50 - 60 °C ) so unable to understand what should be the optimal melting temperature which can be used to amplify the gene segment for sequencing.
I would use the highest annealing temperature that gives a good amount of clean product. Polymerases have an optimum extending temperature and make more errors at too low a temperature so if the annealing temperature is too low the enzyme will still work at the low temperature and may introduce errors near the primer ends on amplification. A high annealing temperature also often means that similar sequences to your gene do not amplify.
I would use the highest annealing temperature that gives a good amount of clean product. Polymerases have an optimum extending temperature and make more errors at too low a temperature so if the annealing temperature is too low the enzyme will still work at the low temperature and may introduce errors near the primer ends on amplification. A high annealing temperature also often means that similar sequences to your gene do not amplify.
Paul is quite right, the higher annealing temperature, the more specific amplification product.
I agree with Paul
- I would say that if you have such a permissive range of specific annealing temp this is a suign of good primer design and optimal target sequence
- To add to the above I would say the higher the better but try and situate the exact temp between Tm-2C and Tm-5C. I say that becuase it sint just about specificity but also efficiency: In other words if you run you products on a gel and notice a single specific band in all of them - good !! - but the intensity of that band decreases then that means the PCR conditions are specific but too stringent; This tends to happen if your annealing temp is above Tm-2C to the Tm
- Thus, use Tm-2C but ideally with such a permissive range of temp try all and run side by side on a gel and pick the annealing temp with the MOST INTENSE band if that falls between Tm-2C to Tm-5C; DO NOT drop below Tm-5C
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