1. Use the buffer supplied with the polymerase.
2. Use the four standard dNTPs (not dUTP or dITP).
3. Some suppliers of dNTPs sell stocks contaminated with dUTP which will cause problems with our thermostable DNA polymerases. To avoid contamination, we suggest NEB dNTPs.
4. Optimize the annealing temperature for the primer.
5. Optimize the magnesium level (2, 4, 6 or 8 mM).
6. Optimize the amount of polymerase (1-2 units per 100 μl reaction for proofreaders; 2-4 units per 100 μl reaction for exo- forms); scale up or down according to reaction volume.
Details on optimization protocols may be found under the Protocols tab.