Q1：Why is there no product or a small amount of product in the final result?？
A1：
1)Template degradation or poor template purity. Use electrophoresis to check template quality and use high-quality templates.
2)The template concentration is too low. Appropriately increase the template usage.
3)The primers are not suitable. Optimize primer design.
4)The annealing temperature is not suitable. Set the annealing temperature gradient and explore the appropriate annealing temperature.
5)The number of cycles is too small. Appropriately increase 5~10 cycles.
6)Insufficient extension time. For complex templates, the extension speed can be appropriately increased to 20~30 s/kb.
Q2：Why do non-specific bands or diffuse bands appear in amplification？
A2：
1)Poor primer specificity. Optimize primers to avoid nonspecific bands and amplification of primer dimers.
2)The primer concentration is too high. Decrease primer concentration.
3)Template excess or poor purity. Reduce the amount of templates and use high-quality templates.
4)The annealing temperature is too low. Appropriately increase the annealing temperature or use the Touchdown PCR program.
5)Too many cycles. Reduce the number of cycles to 25~30 cycles.
