一种大规模筛选单克隆及提取质粒的改进方法

An improved method to select single clones and extract plasmids on a large scale

利用α-互补(X-gal+IPTG)从cDNA文库中筛选特异克隆,以此去除含有未插入片段和插入片段小的克隆。运用展库的方法,通过辅助噬菌体对文库中多个载体的切割,将载体以质粒的形式转化到大肠杆菌中;同时对质粒DNA碱裂解提取方法做了改进,建立了一种简单实用的大量提取质粒DNA的方法。该方法利用特定的蛋白质吸附膜吸附提取过程中的蛋白质,从而去除了使用酚-氯仿抽提蛋白质的复杂过程,减少了质粒DNA的损失,是一次性获得大规模质粒DNA的有效方法。获取的质粒DNA样品在纯度和浓度上都可以满足测序、PCR及Southern杂交等分子生物学实验的要求。

Usingα-complementation(X-gal+IPTG)to choose unique clones from the cDNA library,what can remove the ones with no insert segments or short.According to the method of displaying library,using helper phage to incise a lot of vectors of the library and then transfer the vectors into E.coli in the form of plasmids;at the same time,establishing a simple,convenient method to extract plasmid DNA on a large scale,it is an improved one on the alkaline lysis approach.The key improved point is using specific film to adsorb protein which is abundance in the extraction process and avoiding of the complex steps using phenol-chloroform to extract protein.It reduce the loss of the plasmid DNA and provide an effectively method for extracting a large scale of plasmid DNA at once time.The purity and concentration of plasmid DNA can meet the demand of molecular biology experiments such as PCR?sequencing and Southern hybridization.