PCR buffer optimization with uniform temperature regimen to facilitate automation

Abstract

To facilitate PCR(1,2) reactions in large numbers with uniform conditions, the annealing temperature was fixed and the stringency of the reactions was adjusted by optimizing the ion conditions of the reaction. The buffer system is based primarily on Tris (T), ammonium (N), and potassium (K) to adapt assay conditions to different primer pairs. The TNK buffers have permitted successful screening of a 60,000-clone yeast artificial chromosome (YAC) library with more than 200 primer pairs.