Optimizing depends on application and starting material. Specific recommendations can be found in the protocols. General guidelines are below:
- Perform a temperature gradient to optimize Tm. Unlike with other polymerases, such as Taq, high fidelity amplification can be sensitive to annealing temperatures, where one or two degrees might make the difference between no amplification and successful amplification of a pure product with a high yield. See the data below for an example.
- Use more template. Sample concentration may be too low.
- Optimize enzyme concentration by testing a titration of enzyme in the reaction (0.25-2 units/50 μl reactions)
- Increase number of cycles
- Lengthen extension time to 1min/kb
- Change the extension temperature-some applications (amplifying bisulfite-treated, deaminated or damaged DNA) may benefit from using a higher extension temperature then recommended (72°C v 68°C)