Be sure to use an annealing temperature determined by the nearest neighbor method (or Tm Calculator ). Use fresh high-quality nucleotides. It is important to avoid dNTP mixes that contains dUTP. Use more template. Sample concentration may be too low. Template DNA may be damaged. Use carefully purified template. Lengthen extension time. Increase cycle number. Optimize enzyme concentration. Denaturation temperature may be too low. Optimal denaturation temperature for most templates is 98°C or higher.