Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot3837

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Amplification of cDNA Generated by Reverse Transcription of mRNA

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

In this method, an oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR. Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs. Similarly, the reverse primer used in the subsequent amplification reaction can be gene-specific or general (e.g., random hexamers). To reduce the chance of contamination with exogenous DNAs, prepare and use . . . [Full Text of this Article]


MATERIALS


METHOD


TROUBLESHOOTING


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