Protocol

Dot and Slot Hybridization of Purified RNA

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

Dot blotting of RNA is best carried out using purified preparations of RNA that are denatured with glyoxal or formaldehyde immediately before loading onto a nylon membrane through a vacuum manifold. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H₂O.

MATERIALS

0.1x SSC with 0.1% (w/v) SDS

0.1x SSC with 1% (w/v) SDS

Optional, please see Step 18.

0.5x SSC with 0.1% (w/v) SDS

1x SSC with 0.1% (w/v) SDS

20x SSC

NaOH (10 N)

Prehybridization solution for northern hybridization

Probe, radiolabeled and denatured

Denature just before use as described in Step 2 of Northern Hybridization. . . [Full Text of this Article]

METHOD