Garger S J, Griffith O M, Grill L K
Biochem Biophys Res Commun. 1983 Dec 28;117(3):835-42. doi: 10.1016/0006-291x(83)91672-8.
A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.
本文描述了一种快速制备纯化质粒DNA的方法，并与传统的平衡离心法进行了比较。使用了不连续的两步氯化铯-溴化乙锭梯度，含质粒提取物的起始位置在管底。在固定角度转头中离心5小时，共价闭环质粒DNA可与污染的蛋白质、RNA和染色体DNA分离。通过琼脂糖凝胶电泳和5'-末端标记研究确定，用这种方法纯化的质粒比在均匀梯度中进行48小时平衡离心纯化时受RNA污染的程度要低得多。在两步梯度中纯化的质粒DNA可直接用于限制性内切酶分析和DNA测序。
