Protocol

Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes

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

This protocol describes how to carry out Southern hybridizations at high stringency in phosphate-SDS buffers. Although a wide variety of formats are available, most Southern hybridizations are carried out in heat-sealable bags, roller bottles, or plastic boxes, as described here.

MATERIALS

0.1x SSC

0.1x SSC with 0.1% (w/v) SDS

2x SSC with 0.1% (w/v) SDS

2x SSC with 0.5% (w/v) SDS

6x SSC or

6x SSPE

DNA immobilized on membrane

Phosphate-SDS washing solution 1

Phosphate-SDS washing solution 2

Poly(A) RNA (10 mg/ml) in sterile H₂O

Optional, for hybridization buffers. Prepare solution by dissolving poly(A) RNA in sterile H₂O and . . . [Full Text of this Article]