Protocol

Preparation of Plasmid DNA by Large-scale Boiling Lysis

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

The first 100 words of the full text of this article appear below.

INTRODUCTION

Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA⁺ strains).

MATERIALS

Antibiotic for plasmid selection

Chloramphenicol (34 mg/ml)

Ethanol

Optional, please see Step 5.

Isopropanol

Lysozyme (10 mg/ml)

Prepare the solution fresh in Tris-Cl (pH 8.0).

Restriction endonucleases

Please see Step 4.

Rich medium

STE

STET

TE (pH 8.0)

METHOD

1. Inoculate 30 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic either with a single colony . . . [Full Text of this Article]