Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4055

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Analysis of RNA by Primer Extension

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

Primer extension is used chiefly to map the 5' termini of mRNAs. A preparation of polyadenylated mRNA is first hybridized with an excess of a single-stranded oligodeoxynucleotide primer, which is complementary to the target RNA and radiolabeled at its 5' terminus. Reverse transcriptase is then used to extend the 3' end of the primer. The size of the resulting cDNA, measured by denaturing polyacrylamide gel electrophoresis, is equal to the distance between the 5' end of the priming oligonucleotide and the 5' terminus of the target mRNA.

IMPORTANT:

Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2. . . [Full Text of this Article]


MATERIALS


METHOD


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