小中大第四节 结果分析及注意事项
本实验用PCR技术检测CDK4基因敲入小鼠的表现型成功率为99%。本方法成功率较高，但应注意如下问题：
1．设计引物时，引物的结合位点距离插入基因片段不能太远，否则由于片断太小，使目的基因所占的比例变小，与对照比较时不易拉开距离，效果不好。
2．实验中，常会出现当DNA浓度过高时，反而不能扩增出PCR产物，故应特别注意DNA浓度的控制，具体见本书有关章节。
3．如果PCR未获得扩增结果，需考虑如下问题：
(1) 引物问题：引物可通过电泳检测，有时PCR引物降解则无PCR扩增产物。可使用电泳直接检测引物，如果只有一条带，提示引物降解。
(2) DNA浓度：DNA浓度不能太低或太高。若浓度过高，电泳后，可见加样孔处有浓的DNA，但无特异带，此时需将样品逐级稀释后再扩增。
(3) 若如上均无问题，仍没有PCR产物，应考虑Taq酶活性有无问题。
(4) 反应条件、循环参数是否适宜靶基因的扩增。
邓兴力 王廷华
参 考 文 献
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