Protocol

Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension

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

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP. After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. Because the concentrations of dNTP in the first reaction are nonlimiting, both the amounts and size of cDNA generated are greater than those achieved in standard labeling protocols. IMPORTANT Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated . . . [Full Text of this Article]

MATERIALS

METHOD