Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Maxipreps.

Abstract

In recent years, with the advent of polymerase chain reaction and the development of highly efficient methods of cloning and DNA sequencing, the need to prepare large quantities of plasmid vectors and recombinants has greatly diminished. In consequence, this protocol, at one time in common use, has been largely replaced by faster and easier column-based purification methods. In this protocol, plasmid DNA is purified from the cleared bacterial lysate by centrifugation to equilibrium in CsCl gradients containing ethidium bromide. These gradients have an aesthetic quality that justifies the continued presentation of a protocol that in many other respects is an antique. The typical yield of high-copy-number plasmid vectors or of amplified low-copy-number vectors prepared by this method is ∼3-5 µg of DNA/mL of original bacterial culture. The yield of recombinant plasmids containing inserts of foreign DNA is usually slightly lower, depending on the size and nature of the cloned DNA fragment.