- Be sure to use an annealing temperature determined by the NEB Tm calculator.
- Use fresh high-quality nucleotides. It is important to avoid dNTP mixes that contain 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.
- The DNA template may be GC rich, or supercoiled.
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