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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