DNA电泳(electrophoresis)可能是从事分子生物学研究的小伙伴们进入实验室后做的第一个实验,其重要性不言而喻。尽管DNA电泳实验并不复杂,然后有时候我们的电泳图却并不好看。即便是实验室中的前辈,有时也会遇到这样那样的问题。条带模糊了,不见了,Marker扭曲了,大小不对了等等。本文总结了DNA电泳实验时常见的问题并给出相应的对策,希望对大家有所帮助。
可能原因及对策:
1) DNA降解:避免核酸酶污染;
2) 电泳缓冲液陈旧:电泳缓冲液多次使用后,离子强度降低,pH值上升,缓冲能力减弱,从而影响电泳效果。建议经常更换电泳缓冲液;
3) 所用电泳条件不合适:电泳时电压不应超过20V/cm,温度<30℃;巨大DNA链电泳,温度应<15 ℃;核查所用电泳缓冲液是否有足够的缓冲能力;
4) DNA上样量过多:减少凝胶中DNA上样量;
5) DNA样含盐过高:电泳前通过乙醇沉淀去除过多的盐;
6) 有蛋白污染:电泳前酚抽提去除蛋白;
7) DNA变性:电泳前勿加热,用20mM NaCl缓冲液稀释DNA。
可能原因及对策:
1) DNA的上样量不够:增加DNA的上样量;聚丙烯酰胺凝胶电泳比琼脂糖电泳灵敏度稍高,上样量可适当降低;
2) DNA降解:避免DNA的核酸酶污染;
3) DNA走出凝胶:缩短电泳时间,降低电压,增强凝胶浓度;
4) 对于EB染色的DNA,所用光源不合适:应用短波长(254nm)的紫外光源。
5) 分子大小相近的DNA带不易分辨:增加电泳时间,核准正确的凝胶浓度;
6) DNA 变性:电泳前请勿高温加热DNA链,以20 mM NaCl Buffer稀释DNA;
7) DNA链巨大,常规凝胶电泳不合适:在脉冲凝胶电泳上分析。
可能原因及对策:
1) 配制胶时的缓冲液和电泳缓冲液不是同时配制的。最好是同时配制.电泳时缓冲液高过液面1-2mm即可。
2) 电泳时电压过高,可以在电泳前15分钟用较低电压(3V/cm),等条带出孔后比较整齐了,再调高电压。
3) 上样时尽量慢慢加样,等样品自然沉降后再加电压。
以上内容来源网络,由本人编辑整理。
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