A RAPID AND INEXPENSIVE METHOD FOR PREPARING ESCHERICHIA-COLI PLASMID-DNA

Abstract

A simple, rapid and inexpensive scaled up miniprep procedure for preparing pure Escherichia coli plasmid DNA is described. Bacterial cells were subjected to the boiling procedure and high molecular weight RNA was removed by LiCl-precipitation. Residual RNA and proteins were removed by subsequent treatment with RNase A and proteinase K/SDS respectively, followed by Sephadex G-50 and Sepharose 6B-C1 chromatography. The average yield from a 100 ml over-night bacterial suspension was 100 to 150 ug for pBR-322 DNA, and 25--500 ug for SP-6 derived recombinant plasmids. In addition, the described "scaled up" preparation doses not require CsCl-ethidium bromide centrifugation; pure plasmid DNA can be prepared within 1 to 2 days.