Mukhopadhyay M, Mandal N C
Anal Biochem. 1983 Sep;133(2):265-70. doi: 10.1016/0003-2697(83)90080-5.
A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.
本文描述了一种从细菌中制备纯质粒DNA的非常简单且成本低廉的方法。在该方法中，在含有833微克/毫升溴化乙锭的情况下通过溶菌酶诱导形成原生质球，然后用Brij 58和脱氧胆酸钠的混合物裂解原生质球，并将裂解物在48,000g下离心25分钟，从而使约99.9%的总染色体DNA沉淀。从含有质粒DNA的上清液中，通过苯酚抽提去除蛋白质，并通过氯化钙沉淀去除大部分RNA，最后通过核糖核酸酶处理去除少量残留RNA。通过该方法从1升扩增培养物中获得的pBR322 DNA的平均产量为2至2.5毫克，并且制备物纯度很高，仅含有约0.005%的总产率作为染色体DNA污染物。此外，通过该方法制备的质粒DNA的底物活性和转化能力不受影响。
