在分子克隆的试验中，如何快速检测目标序列已成功插入到质粒一直是个值得讨论的问题。BioEngX以前提到过用colony PCR或是质粒测序的方式（colony PCR ，戳）检测阳性质粒（positive plasmid）。colony PCR原理很简单，就是直接使用菌落作为PCR母板，尝试反应扩增目标序列，如果有目标条带，则证明序列已成功插入。那么，还有没有比colony PCR还要快的检测方法？自然是有的—-colony cracking.
Colony cracking，小编愿意称它为菌落破裂法，原理简单，直接暴力，适合于快速检查大量菌落。把菌落直接裂解，将裂解液跑过琼脂胶，通过观察携带目标片段的质粒和普通质粒之间移动距离的差别，来判断对应菌落是否携带阳性质粒。显然如果插入片段越大，阳性质粒和普通质粒在移动距离上就会差别越大，也就越容易看出区别。
配置裂解液
1X裂解液组成成分
100mM NaOH, 60mM KCl, 5mM EDTA, 0.5% SDS
方法
- 菌落生长到直径2 mm左右（菌落太小会导致所含质粒的量不足，不易于显示于琼脂胶上）。
- 挖一半菌落，投于20 uL 裂解液中。
- 55°C 培养20 min。
- 置于冰上2 min。
- 最大速度离心 3 min。
- 裂解液加loading buffer后，进行电泳。
注意
- 检测质粒最好是高复制量的质粒，否则菌落携带质粒太少，跑胶后的质粒DNA信号和细胞基因组DNA信号无法区别。
- colony cracking不是一个能准确检测插入片段大小的方法，只能检测是否有片段插入到了质粒中。
- 当有大量菌落需要被检测，而且时间有限的时候，建议用colony cracking；如果只需检测一两个菌落，那么简单的colony PCR或是提取质粒送测序则更为实用。
- 细胞裂解后自身基因组DNA也会被释放出来，因此在琼脂胶中出现很多背景信号也是正常，有可能会影响观察。
Reference
Plasmid detection and sizing in single colony lysates. WM Barnes. Science 28 January 1977: Vol. 195. no. 4276, pp. 393 – 394.
你尝试过colony cracking这种方法吗？欢迎留言讨论~~
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