Protocol

Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous Gradients

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

Solutions containing plasmid DNA are adjusted to a density of 1.55 g/ml with solid CsCl. The intercalating dye, ethidium bromide, which binds differentially to closed circular and linear DNAs, is then added to a concentration of 200 mu;g/ml. During centrifugation to equilibrium, the closed circular DNA and linear DNAs form bands at different densities.

MATERIALS

Crude plasmid preparation

Use material from either Step 16 of Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation, Step 18 of Preparation of Plasmid DNA by Large-scale . . . [Full Text of this Article]