DNA polymerases possessing high fidelity due to a proofreading exonuclease moiety are more exacting in their use than their exo- derivatives or DNA polymerases without a proofreading exonuclease, such as Taq DNA Polymerase. Also, differences in the Km (DNA) possessed by different DNA polymerases can change the effective primer annealing temperature. We suggest reviewing the general guidelines accompanying the polymerase and adhering to the following suggestions: 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 (0.25-2 units per reaction for proofreaders; 2-4 units per reaction for exo- forms); scale up or down according to reaction volume. Details on optimization protocols may be found under the general Polymerase Technical Reference page.