将外源质粒导入大肠杆菌的操作被称之为转化。除了最传统的化学转化(Chemical Transformation)外,还有电穿孔转化(Electroporation)的方法。本期我们聊聊两种转化的优劣和基本原理,后续还有更多关于感受态细胞(competent cell)和不同细胞系选择的内容。细菌细胞转化是非常常见而且简单的分子实验,只要稍加实践练习,就很容易掌握。如果你已经是转化高手,那么这个系列的文章就权当温习,欢迎评论并且分享自己的转化经验;如果你是新手,不妨来看看~~~
电转化(Electroporation)
电转化原理十分简单,当电流穿过细胞的时候,会在细胞膜上形成临时的孔洞,那么携带负电的质粒DNA在这个电势场中会被移动,而后恰好穿过孔洞,进入细胞。这个过程和电泳类似,DNA分子移动来移动去,最后掉进了细胞里。
化学转化(Chemical Transformation)
正常的大肠杆菌细胞与CaCl2 溶液混合后,许许多多的Ca+离子附着在细胞膜表面,带正电的钙离子们有助于引导和辅助带负电的DNA质粒与细胞膜结合。当质粒DNA穿膜进入细胞的时候,正电钙离子也很好的掩盖了DNA的负电性,使得通过憎水性磷脂层的过程更加容易。使质粒进入细胞的方法是短暂42°C的热击。
小编做化学转化的一般方法是:(细胞+质粒DNA)混合 >>> 冰上培养20 min >>> 42°C热水浴30s >>> 冰上培养2 min >>> 细胞与250 uL LB或S.O.C 混合培养1h >>> 50 uL培养液涂板 >>> 细胞长长长,37°C。
两种方法都十分简单,基本上每家生产感受态细胞的厂商都会附上一份对于他们的细胞可以用的转化方法的操作流程。小编自己觉得,电穿孔转化的效率会比化学转化要高一些,电转化更节省时间,同时呢,电转化会需要特别的设备,比如带电机的小容器(附图见文后),所以成本会比化学转化高一些。尽管化学转化有时候效率不高,但通过增加DNA的量或者选择不同的感受态细胞系,我们是可以做到高效率转化的。小编平常一直使用化学转化的方法,效率也还不错,诶~,就是个熟能生巧的过程!
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