相信大家在做PCR反应时总能遇到这样或是那样的问题，但多数都可以归为两个主要原因：
- 基因模板的扩增太少（amplification）;
- 非目标基因扩增太多。
但是大家可以使用添加剂来解决这些问题。通常添加剂的作用有两方面：
- 降低基因的二级结构（secondary structure）;
- 降低非特异性的启动（priming）。
上一期小编给大家简单介绍了可以减低基因二级结构的添加剂，今天再接着介绍一下能够降低非特异性启动的添加剂以及其他一些常见的添加剂。
甲酰胺（Formamide）
甲酰胺是一种常用的有机PCR添加剂。它可以和DNA中的大沟（major groove）和小沟（minor groove）相结合，从而降低母版DNA双螺旋的稳定性并且减低DNA的解链温度（melting temperature）。甲酰胺在PCR实验中使用的浓度通常在1%-5%。
四甲基氯化铵（Tetramethyl ammonium chloride，TMAC）
四甲基氯化铵可以增加杂化的特异性（hybridization specificity）并且提高DNA的解链温度。因此，TMAC 可以去除非特异性的启动，并且减少DNA和RNA的错误结合。如果大家在PCR反应中使用简并引物（degenerate primers）时，记得可以添加TMAC，它常用的使用浓度为15-100mM。
除了以上提到的两大类添加剂，PCR反应中还有许多常见的添加剂，虽然作用不同，但也至关重要。
镁离子（Magnesium ion）
镁离子是聚合酶不可缺少的一个辅因子（cofactor），也就是说如果没有镁离子的话，聚合酶是没有活性的。但是，过多的镁离子同样也会影响聚合酶的工作效率。每种PCR反应中镁离子的浓度都不尽相同。螯合剂（Chelating agents，例如EDTA 或是 citrate）, dNTP 的浓度和蛋白质都会影响镁离子的浓度。所以，如果你的PCR实验有问题，可以尝试改变不同的镁离子浓度，例如从1.0到4.0mM，中间间隔 0.5–1mM 就可以。
值得注意的是，多次冻融循环能够导致氯化镁溶液（magnesium chloride）的浓度分层。所以大家在每次使用之前一定要完全溶解，并且混合均匀之后再使用。
牛血清白蛋白（Bovine albumin，BSA）
在分子化学实验中，牛血清白蛋白是很常见的添加剂，尤其在限制酶酶切和PCR的实验中。在PCR反应中，BSA 对于减少污染物，例如酚类化合物（phenolic compounds）有一定帮助。而且也有说法是它能减少反应物附着在试管壁的情况。在PCR反应中，通常BSA添加的浓度可达到0.8 mg/ml。
欢迎大家在平台留言交流更多关于PCR的小技巧！
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