Plasmid Purification Made Easy

Nucleic acid purification procedures have enjoyed a long history, beginning with the first isolation of "nuclein" in 1869 by Freidrich Meischer. In 1958, Matthew Meselson and Frank Stahl demonstrated semiconservative replication of DNA using density-gradient centrifugation, an isolation method that became the cornerstone of recombinant DNA technology in the '70s and '80s. The technology was effective--producing high-quality DNA--but it was not efficient.

The classical method of isolating plasmid DNA from bacterial cells, called the alkaline lysis procedure, relies on density-gradient centrifugation.¹ After incubating cells in an isotonic buffer, the cells are lysed in an alkaline solution containing detergent, which also denatures the DNA. Addition of a neutralizing solution--usually buffered potassium acetate--causes proteins and genomic DNA to crash out of solution, while the smaller plasmid DNA simply renatures, remaining in solution. The plasmid DNA is either ethanol precipitated or phenol-chloroform extracted, and then purified by cesium chloride-ethidium bromide density-gradient ultracentrifugation....