Paul Bonnie, Cloninger Cheri, Felton Marilyn, Khachatoorian Ronik, Metzenberg Stan
Department of Biology, California State University, Northridge, CA 91330-8303, USA.
Anal Biochem. 2008 Jun 15;377(2):218-22. doi: 10.1016/j.ab.2008.03.005. Epub 2008 Mar 7.
We describe a simple method of isolating plasmid DNA directly from Escherichia coli culture medium by addition of lithium acetate and Sodium dodecyl sulphate, followed by centrifugation and alcohol precipitation. The plasmid is sufficiently pure that it can be used in many enzyme-based reactions, including DNA sequencing and restriction analysis. Chromosomal DNA contamination is significantly reduced by pretreatment of the culture with DNase I, suggesting that much of the contaminant is associated with permeable dead cells. Chromosomal DNA contaminant can also be selectively denatured without damage to the supercoiled plasmid by alkaline denaturation in an arginine buffer or heat treatment in the presence of urea or N,N-dimethylformamide.
我们描述了一种直接从大肠杆菌培养基中分离质粒DNA的简单方法，即加入醋酸锂和十二烷基硫酸钠，然后进行离心和乙醇沉淀。所得到的质粒纯度足够高，可用于许多基于酶的反应，包括DNA测序和限制性分析。用DNase I对培养物进行预处理可显著减少染色体DNA污染，这表明大部分污染物与可渗透的死细胞有关。通过在精氨酸缓冲液中进行碱性变性或在尿素或N，N-二甲基甲酰胺存在下进行热处理，也可以选择性地使染色体DNA污染物变性而不损害超螺旋质粒。
