Protocol

Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I

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

Probes of high specific activities can be obtained using the Klenow fragment of E. coli DNA polymerase I to catalyze synthesis of a strand of DNA complementary to a synthetic oligonucleotide. A short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment of E. coli DNA polymerase I to incorporate [-³²P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation, followed by electrophoresis through a polyacrylamide gel under denaturing conditions. With this method, it is . . . [Full Text of this Article]

MATERIALS

METHOD