Kieser T
Plasmid. 1984 Jul;12(1):19-36. doi: 10.1016/0147-619x(84)90063-5.
Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids.
基于对影响质粒产量和纯度因素的系统研究结果，开发了一种适用于从淡紫链霉菌和大肠杆菌中快速筛选和分离共价闭合环状DNA的方法。该方法包括用溶菌酶处理细菌使其裂解，并结合高温下DNA的碱性变性。通过加入少量苯酚/氯仿实现共价闭合环状DNA的复性以及单链DNA与蛋白质的沉淀。筛选过程仅使用单个试管，裂解后约30分钟即可通过琼脂糖凝胶电泳对样品进行分析。通过用异丙醇和精胺连续沉淀，随后用乙醇萃取，可去除苯酚并进一步纯化质粒制剂，约2小时后得到适合限制性内切酶消化、连接以及转化淡紫链霉菌原生质体或感受态大肠杆菌细胞的样品。该方法的所有步骤都有详细解释，并提供了参数变化影响的相关信息。这应有助于实验者获得可重复的结果，并且在该方法需要适用于新菌株或质粒时可能会很有用。
