Welter C, Meese E, Blin N
Institute of Human Genetics, University of the Saar, Homburg, FRG.
Mol Biol Rep. 1988;13(2):117-20. doi: 10.1007/BF00539059.
A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5'-end labeling, gel retention assays, and various types of hybridization.
本文描述了一种便捷的阶跃梯度（氯化铯/溴化乙锭）方法改进。这种快速方法能够在约5小时内从污染的蛋白质、RNA和染色体DNA中分离出共价闭合环状DNA。可对真核细胞器（线粒体）的环状DNA进行大规模制备。该方案对线粒体采用细胞器沉淀/氯化钠-肌氨酸孵育步骤，随后进行氯化铯阶跃梯度分离，其产量与传统方法相当。所得到的DNA纯度足以用于限制性内切酶分析、亚克隆、5'端标记、凝胶滞留试验及各种杂交类型。
