Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4054

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Ribonuclease Protection: Mapping RNA with Ribonuclease and Radiolabeled RNA Probes

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

Preparations of RNA containing an mRNA of interest are hybridized to a radiolabeled single-stranded RNA probe. At the end of the reaction, a mixture of RNase A and RNase T1 is used to degrade unhybridized regions of the probe, and the surviving molecules are then separated by denaturing gel electrophoresis and visualized by autoradiography. The method can be used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O.


MATERIALS

recipe 10x Transcription . . . [Full Text of this Article]


METHOD


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