Protocol

Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates

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

A synthetic oligonucleotide annealed to single-stranded DNA derived from a recombinant bacteriophage M13 or phagemid template is used to prime the synthesis of complementary radiolabeled DNA. Synthesis is catalyzed by the Klenow fragment of E. coli DNA polymerase I, which extends the annealed primer for various distances along the single-stranded template DNA. The products of the reaction, which are heterogeneous both in length and in the amount of incorporated radiolabeled dNTPs, are digested with a restriction enzyme to create double-stranded DNA fragments of uniform length, which are subsequently purified by agarose gel electrophoresis.

The oligonucleotide primer is usually complementary to . . . [Full Text of this Article]

MATERIALS

METHOD