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质粒提取试剂盒中各溶液配方及作用

发布: 2016-02-12 1,534阅读 199评论

提质粒是分子生物学中最基本，最easy的实验。完全不需要借助大脑，直接照着提取试剂盒中的操作说明傻瓜操作即可。小编一直认为应该发明一个机器人在实验室中操作这些简单却耗时的实验。尽管操作简单，提质粒却也是一个比较重要的步骤，提出质粒的质量和数量将直接决定着后续实验室的成败。当我们按照试剂盒中操作说明进行操作时，我们会看到P1，P2，N3, PE，EB等不同的溶液（不同试剂盒可能标识不同）。那么大家是否知道每瓶溶液中装的什么？它们在质粒提取过程中的作用又是怎样的？提质粒很简单，但其背后的学问却并不简单哦。

质粒提取采用的是强碱裂解法（alkaline lysis），这个方法是由Birnboim和Doly在1979年发明的，在SCI上原著文章的引用量至今为止已达到12886。有关碱裂解法的原理可以见BioEngX历史文章提取质粒DNA和基因组DNA的区别。那么我们现在来看一下溶液 P1 ， P2， N3, PE, EB都是什么。

溶液1（P1）

组分浓度  25 mM Tris-HCl（pH8.0），10 mM EDTA，50 mM 葡糖糖（Glucose）

溶液1的主要作用是将菌体沉淀悬浮起来。25mM Tris-HCl是缓冲溶液，保证反应体系的pH恒定。EDTA是金属离子螯合剂，10 mM EDTA的作用是与微生物体内的金属离子相互结合，抑制DNase的活性。50 mM葡萄糖的作用是增加溶液的粘度，保证菌体悬浮，延缓菌体沉降的时间。溶液1在使用前通常要加入RNA酶（RNase A），RNA酶的作用很清楚，降解掉溶液中的RNA。由于RNA酶属于蛋白质，蛋白质稳定性很差，所以加了RNase A的溶液1需要低温4oC保存。

溶液2（P2）

组份浓度 250 mM NaOH，1％（W/V）SDS（十二烷基硫酸钠）

溶液2的主要作用是细胞裂解。溶液1将细胞悬浮后，就需要添加溶液2了，溶液2的作用就是对细胞进行裂解。溶液2只包括两种成分：氢氧化钠和SDS。真正起到到细胞裂解作用的是氢氧化钠，这也是为什么这个方法会被叫做碱裂解法。氢氧化钠会破坏细胞膜的结构，使之发生bilayer（双层膜）结构向micelle（微囊）结构的相变化，从而导致细胞的裂解。SDS的作用将在下一步提到。添加溶液2后需要注意的一个是时间一定不能过长，另一个是不可以剧烈震动离心管。时间过长以及剧烈震动都将导致氢氧化钠破坏基因组DNA，断裂的基因组DNA碎片将会与相似大小质粒一同被抽提，污染样品。

溶液3（N3）

组份浓度 3M 醋酸钾（potassium acetate），5M 醋酸

溶液3的主要作用是中和第二步加入的强碱以及清除掉蛋白质等细胞内的杂质。N是neutralized的缩写。强碱溶液氢氧化钠长时间与DNA基础会破坏其结构，因此需要进行中和。中和氢氧化钠，5 M醋酸就足够了，为何又要加入醋酸钾呢？做过质粒提取实验的同学应该记得，在加入溶液N3后，会有大量沉淀出现。这些沉淀其实醋酸钾与溶液2中的SDS相互作用，反应生成的十二烷基硫酸钾（potassium dodecylsulfate, PDS）。加入溶液N3后，SDS遇到钾离子，生成PDS不溶于水，会迅速发生沉淀。

那么溶液3的加入是如何清除掉蛋白质，基因组DNA等杂质的呢？道理是，溶液2中加入的十二烷基硫酸钠(SDS)很容易与蛋白质结合，平均两个氨基酸就会结合一个SDS分子，二者结合会形成SDS2多肽复合体，这样变性蛋白质将溶解在溶液中，从而使得多肽链更好地与基因组DNA缠绕。加入溶液3后，由于十二烷基硫酸盐与变性蛋白质结合成复合体，十二烷基硫酸钾的沉淀就将变性的蛋白质一起沉淀，同时变性的蛋白质与基因组DNA缠绕，结果也大部分被共沉淀了。而高离子浓度的溶液又加速了这一沉淀过程。此外，溶液被中和后变性蛋白质表面电荷减少,也可以促进变性蛋白质沉淀。

溶液PE （wash buffer）

组分浓度10 mM Tris-HCl （pH 7.5），  80 % 乙醇（Ethanol）

Wash buffer的作用主要是清洗掉多余的盐离子。试剂盒中都是利用硅胶柱进行DNA提取的，有关硅胶柱提取DNA原理可见BioEngX历史文章”提纯你的DNA样本，很急，很关键！“。在DNA与硅胶柱吸附后，需要利用Wash buffer清洗掉多余的盐离子。Wash buffer中含有高浓度的乙醇，由于乙醇会影响后续的酶切或测序反应，在洗脱DNA前必须要在离心机内”空甩”柱子，完全清除掉乙醇。

溶液EB（Elution buffer）

组分浓度10 mM Tris-HCl （pH 7.5）

EB的作用就是洗脱硅胶柱上的DNA样品。Elution buffer中不能含有过高浓度的盐离子，因为在高盐环境中，盐阳离子会打破硅胶负氧根和水之间的氢键，使得整体的硅胶携带正电，会吸引携带负电的DNA分子。另外，加热过的洗脱液（<50°C）可以加速DNA从硅胶膜上脱离下来。另外，离心前保持EB缓冲液在膜上停留时间长一些，将更有利于DNA的洗脱。

不同公司的质粒提取试剂盒中溶液成分可能有所差异，比如P1中可以去除掉葡萄糖，溶液PE甚至可以直接用水来代替等等。质粒提取实验是分子生物学中的基本且重要的实验，尽管实验本身简单，但其原理还是有些复杂的。小编相信，知其然亦要知其所以然，清楚实验的原理，当实验出现问题之时，会能够更容易找到原因并给予纠正。

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