Protocol

Hybridization of Bacterial DNA on Filters

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 procedures to hybridize DNA from transformed colonies immobilized on filters with radiolabeled probes and to recover from a master plate the corresponding colonies that hybridize specifically to the probe. The method is based on the procedure published by Grunstein and Hogness (1975).

MATERIALS

³²P-labeled double-stranded DNA probe or Synthetic oligonucleotide probes

Filter Wash Solution 1

Filter Wash Solution 2

Filter Wash Solution 3

Filters with immobilized DNA from transformed colonies

Use filters prepared as described in Lysing Colonies and Binding of DNA to Filters.

Formamide

Prehybridization/hybridization solution (for Plaque/Colony Lifts)

Prewashing solution (1-32)

Rich medium containing the . . . [Full Text of this Article]

METHOD