Nicole Nichols, PhD:
Most primer design tools, or Oligo manufacturers, will calculate melting temperatures or Tm's, based on a standard set of conditions. The Tm can then be adjusted to get an annealing temperature, or Ta, for your particular reaction.
There are options to modify the standard conditions so that it reflects the actual PCR buffer being used. But in our experience, most users don't alter these values. And in truth, for many cases, there are a wide range of temperatures that will support robust amplification. So getting an appropriate Ta isn't that difficult for these situations.
In other situations, due to primer design issues or template complexities, finding an appropriate Ta is actually quite difficult. In addition, not all enzymes are like Taq and some require a different modification to get from an appropriate melting temperature, to a useful Ta.
For Taq-based products, the typical recommendation is to subtract five degrees from your Tm, to get a Ta useful for your experiment.
On the other hand, with our Q5 high fidelity DNA polymerase, this enzyme prefers and performs better with higher annealing temperatures. Because higher Ta's contribute to Q5's robustness on difficult amplicons and specificity, it's an important detail that shouldn't be overlooked.
Since standard methods for calculating Tm's and adjusting them to get to a Ta yield results that are too low for Q5. We've developed a NEB online tool, the NEB Tm Calculator, that can be used for any of our polymerases to get an appropriate Ta for your experiment.
Our Tm calculator takes actual buffer conditions into account when calculating Tm's for your primer pair by asking you not just for your primer sequences, but for the enzyme and buffer that you intend to use for your reaction. And because you can enter your primers as a pair, you will get not just the melting temperature for each of the primers, but you'll get an appropriate adjustment from the melting temperature to the actual Ta or annealing temperature that you should use for your experiment for the best results.
Using the NEB Tm Calculator is an important step to ensuring that you have specific and robust results from your reactions every time.
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