FACTORS AFFECTING THE ISOLATION OF CCC DNA FROM STREPTOMYCES-LIVIDANS AND ESCHERICHIA-COLI
FACTORS AFFECTING THE ISOLATION OF CCC DNA FROM STREPTOMYCES-LIVIDANS AND ESCHERICHIA-COLI
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DOI：
10.1016/0147-619x(84)90063-5
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发表时间：
1984-01-01
期刊：
影响因子：
2.6
通讯作者：
KIESER, T
中科院分区：
文献类型：
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作者：
KIESER, T
在系统研究影响胞质产量和纯度因素的基础上，提出了一种快速筛选和分离S. lividans和E.大肠杆菌中进行了筛选。该方法包括裂解溶菌酶处理的细菌结合高温下DNA的碱变性。CCC DNA的复性和单链DNA与蛋白质的沉淀通过加入最少量的苯酚/氯仿来实现。筛选程序仅使用一个试管，样品可以在裂解后约30分钟通过琼脂糖凝胶电泳进行分析。通过用异丙醇和精胺连续沉淀，随后用乙醇提取，产生适合于限制性内切酶消化、连接和转化S. lividans原生质体或感受态E. coli细胞培养约2 h。该过程的所有步骤都详细解释了有关更改参数的影响的信息。这将有助于实验者获得可重复的结果，如果该方法必须适应新的菌株或质粒，这可能是有用的。
Based on the results of a systematic study of factors affecting plasmic yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular (CCC) DNA from S. lividans and E. 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, folllowed 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.