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琼脂糖凝胶电泳，快来看各种细节~

发布: 2017-04-26 911阅读 199评论

凝胶电泳（gel electrophoresis）算得上是分子生物实验中最常见的技术了。在实验室里，我们常常称之为跑胶。特别是用来分离不同长度DNA片段的琼脂糖胶，由于原理简单、操作容易，更是被频繁使用。小编和琼脂糖胶打交道也有几年了，觉得跑胶确实很简单，同时整套操作中的细节也是非常多的。如何跑出完美的胶，做好这些细节很重要。

琼脂糖和琼脂糖胶

一种从海带中提取的长链多糖。每个重复单元是由两个单糖组成的（如下图）

 

当琼脂糖融电解缓冲液加热后，是自由的零散的多糖链。随着温度的降低，糖链和糖链之间会自然形成稳定的螺旋结构，随后再聚合，形成网状结构（图1）。在电镜下，我们确实可以看到形成的那种网状结构（图2）。可以想象，不同大小的DNA片段在通过这些网的时候，会遇到不同的阻力。大块头的DNA片段经过时，阻力大，速度就会慢些。

琼脂糖胶的浓度一般因实验而定，0.5%-2%之间都是可以的。浓度越高，使用的琼脂糖越多，形成的网格也就越密。一般情况下小编使用的是1%的琼脂糖胶，即1g 琼脂糖溶于100 ml 电泳缓冲液。每次加热琼脂糖水溶物时，都要考虑加热过程中的水分蒸发流失，否则做出的胶浓度是比预定浓度高的。

 

图1：从琼脂糖到琼脂糖胶的结构变化。

 

图2：电镜下的琼脂糖胶

 

倒胶

加热过后的琼脂糖胶，需要加入可以显色DNA的荧光染料。这样DNA与染料结合物就可以在UV下被激发显出荧光条带。最常见的染料是溴化乙锭（ethidium bromide），在胶中浓度为0.5 µg/ml即可。

电泳缓冲液

最常见的两种是TAE（Tris-acetate-EDTA）和TBE（Tris-borate-EDTA）。缓冲液提供稳定的pH和充足的离子含量。多次使用的缓冲液，导电能力下降，pH上升，可能会产生模糊不规则的DNA条带甚至是不规则移动。所以呀，经常换换电泳槽里的缓冲液是十分重要的。

上样缓冲液

用于混合DNA样本的缓冲液，一般含有沉降物质（如，甘油）和追踪染料（如，溴酚蓝 【bromophenol blue】 和二甲苯青 【xylene cyanol】）。在1%的胶中，溴酚蓝的移动速度和300bp的双链DNA片段相似，二甲苯青的移动速度类似于4000bp的双链DNA。查看追踪染料的移动距离，可以帮助我们了解跑胶大致跑到什么程度了，防止了过度跑胶。如图3，就是常见的追踪染料在琼脂糖胶中的移动速度。

图3：追踪染料的移动速度

 

电压

电压的选择，取决于两个电极之间的距离。电压过高，电流流通量会变大，那么很容易在电泳槽中产生热量，最终可导致胶的变型甚至融化，影响条带的清晰度。一般遵循5V/cm 的原则（距离是两个电极之间的距离，并非仅仅是胶的长度），保证了电压不会过大。跑胶时间要根据自己的习惯和实验室所用电泳槽的大小来决定。

小编能够想到的细节就这些，想必童鞋们也都有各自关于电泳的心得体会，欢迎留言讨论，指正偏颇。做好细节，就能跑出好胶。

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