Protocol

A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissues

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

This protocol, a variation of the method described in Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-Chloroform Extraction, involves lysis of cells in a monophasic solution of guanidine isothiocyanate and phenol. Addition of chloroform generates a second (organic) phase into which DNA and proteins are extracted, leaving RNA in the aqueous supernatant. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 µg/mg starting tissue or 5-10 µg/10⁶ cells. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H₂O.

MATERIALS

Chloroform

Ethanol

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