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Table 2 The UM-PCR procedure (“Two Rounds Mode”)

From: Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments

  Temperature (°C) Time
  94 5 min
94 40s
annealing temperature of primer 1 20s
annealing temperature of primer 2 20s
annealing temperature of primer 3 20s
annealing temperature of primer 4 20s
annealing temperature of primer 5 20s
72 30s
94 40s
70 50s
  72 10 min
  1. In the first round (the first three cycles), annealing temperatures that are optimized in accordance with the Tm of normal SSR primers are sorted in descending order. Annealing temperatures may be different in various combinations of U-primers.