Protocol

Preparation of Plasmid DNA by Lysis with SDS

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

Large (>15 kb), closed circular plasmids are prepared (albeit inefficiently and in small yield) by lysing bacteria with SDS.

MATERIALS

Antibiotic for plasmid selection

Chloramphenicol (34 mg/ml)

Chloroform

Optional, please see Step 7.

EDTA (0.5 M, pH 8.0)

Ethanol

Optional, please see Step 5.

Lysozyme (10 mg/ml)

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

NaCl (5 M)

Store the solution of NaCl at 4°C.

Phenol:chloroform (1:1, v/v)

Restriction endonucleases

Please see Step 4.

Rich medium

SDS (10% w/v)

STE, ice cold

TE (pH 8.0)

Tris-sucrose

METHOD

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