Q1：扩增无产物或产物量少？

A1：

1) 模板降解或模板纯度差。电泳检测模板质量，使用高质量模板；

2) 模板浓度过低。适当增加模板用量；

3) 引物不合适。优化引物设计；

4) 退火温度不合适。设置退火温度梯度，摸索合适的退火温度；

5) 循环数过少。适当增加5~10个循环；

6) 延伸时间不足。复杂模板可适当增加延伸速度至20~30 s/kb。

Q2：扩增出现非特异条带或弥散带？

A2：

1) 引物特异性差。优化引物，避免非特异性条带以及引物二聚体的扩增；

2) 引物浓度过高。降低引物浓度；

3) 模板过量或纯度差。减少模板量，使用高质量模板；

4) 退火温度过低。适当提高退火温度或使用Touchdown PCR程序；

5) 循环数过多。减少循环数至25~30 cycles。

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