Wu G J, Cannon R E
Experientia. 1985 Nov 15;41(11):1488-90. doi: 10.1007/BF01950053.
A reproducible and economical procedure for obtaining a large and quantitative yield of highly purified covalently closed circular plasmid DNA is described. The procedure departs in several ways from more commonly used methods. These are a) avoidance of the use of CsCl, ethidium bromide and ultracentrifuge, b) enrichment of the plasmid DNA by selective denaturation of chromosomal DNA with an alkaline-SDS solution, c) enrichment of covalently closed circular plasmid DNA by extraction with acid-phenol, and d) removal of small degraded RNA fragments by molecular sieve chromatography after digestion with RNase A. The plasmid DNA prepared by this new procedure is free of contaminants and has been used for DNA sequencing, in vitro transcription, transformation and in vitro mutagenesis.
本文描述了一种可重复且经济的方法，用于大量获得高纯度的共价闭合环状质粒DNA。该方法在几个方面与更常用的方法不同。这些方面包括：a) 避免使用CsCl、溴化乙锭和超速离心机；b) 用碱性SDS溶液选择性变性染色体DNA来富集质粒DNA；c) 用酸酚抽提来富集共价闭合环状质粒DNA；d) 用RNase A消化后通过分子筛色谱去除小的降解RNA片段。通过这种新方法制备的质粒DNA没有污染物，已用于DNA测序、体外转录、转化和体外诱变。
