A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

• Published in: Analytical biochemistry Vol. 133; no. 2; p. 265
• Main Authors: Mukhopadhyay, M, Mandal, N C
• Format: Journal Article
• Language: English
• Published: United States 01-01-1983
• Subjects: Calcium Chloride; Chromosomes, Bacterial - analysis; DNA, Bacterial - isolation & purification; Escherichia coli - analysis; Ethidium; Methods; Plasmids; Ribonucleases
• ISSN: 0003-2697
• 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.