Protocol

Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation

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

Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with alkali and SDS.

MATERIALS

Alkaline lysis solution I

For preparations of plasmid DNA that are to be subjected to further purification by chromatography (please see Purification of Plasmid DNA by Chromatography), sterile Alkaline lysis solution I may be supplemented just before use with the appropriate volume of 20 mg/ml DNase-free RNase A (pancreatic RNase) to give a final concentration of 100 µg/ml

Alkaline lysis solution II

Alkaline lysis solution III

Antibiotic for plasmid selection

Chloramphenicol (34 mg/ml)

Ethanol

Optional, please see Step 5.

Isopropanol

Lysozyme (10 mg/ml)

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