Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4060

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Transfer and Fixation of Denatured RNA to Membranes

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

This protocol describes the transfer of RNA from agarose gels to neutral or positively charged nylon membranes, using upward capillary flow of neutral or alkaline buffers. RNA becomes covalently fixed to positively charged nylon membranes during transfer in alkaline buffers. However, treatment by UV irradiation or heating is required to fix RNA to neutral membranes. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O.


MATERIALS

recipe 0.2x SSC with 1% (w/v) SDS

recipe 20x SSC

recipe caution Ammonium acetate (0.1 M) with 0.5 µg/ml ethidium bromide

Optional, please see Step 13.

recipe caution Methylene blue solution

RNA Transfer buffer

For alkaline transfers . . . [Full Text of this Article]


METHOD


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