Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4013

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Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligonucleotides

Joseph Sambrook and David W. Russell

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

Plaques formed by M13 bacteriophages or bacterial colonies transformed by plasmids carrying specific mutations can be detected by hybridization, using a radiolabeled oligonucleotide that forms a perfect duplex with the mutant sequence. Hybridization is carried out under conditions of low stringency that allow the radiolabeled oligonucleotide to anneal to both mutant and wild-type DNAs. A hybrid between the radiolabeled oligonucleotide and the wild-type sequence will contain one or more mismatched base pairs, whereas the hybrid formed between the newly created mutant and the oligonucleotide probe will be perfectly matched. These two types of hybrids usually differ in their thermal stabilities, . . . [Full Text of this Article]


MATERIALS


METHOD


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