I am doing a PCR, and I checked the annealing temperature of my oligos in the NEB Tm calculator. When I selected Q5 polymerase, it said the annealing temperature was 58 degrees celsius, but when I chose Taq polymerase, it showed the annealing temp to be 56 degrees Celsius. How is the annealing temp differs from using another polymerase?
What NEB tm calculator provides is to estimate the proper annealing temperature, that is often affect by salt concentration (e.g. MgCl2) of buffer based on each kit (e.g. Q5, phusion, and etc.), while other online available tm calculator used default salt concentration to estimate.
For further reading: Article Impact of DNA Polymerases and Their Buffer Systems on Quanti...
According to https://tmcalculator.neb.com/#!/help :
Why is the primer Tm (or annealing temperature) different from other Tm calculators?
The NEB Tm calculator is designed to take into account the buffer conditions of the amplification reaction based on the selected NEB polymerase. Many Tm calculators do not, relying instead on a default salt concentration. The annealing temperature for each polymerase is based on empirical observations of efficiency. The optimal annealing temperature for high fidelity hot start DNA polymerases like Q5 may differ significantly from that of Taq-based polymerases.
Hi there,
In theory annealing temperature depends only on the composition of the sequences you want to hybridize. The nature of the polymerase is basically of no effect. However the composition of the PCR mixture (salt, pH...) will affect the strentgh (and therefore the stability) of the interaction between the 2 strands. As the composition of the PCR reaction does vary with the polymerase used, it is improperly said that the hybridization is dependent on the enzyme.
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