Phenol/SDS Method for Plant RNA Preparation


Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.3
DOI:  10.1002/0471142727.mb0403s09
Online Posting Date:  May, 2001
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Abstract

The method described here can be used to prepare RNA from a variety of eukaryotic tissues. The critical factor in isolating RNA from eukaryotic tissues is inactivating the endogenous RNase and preventing introduction of RNase from external sources. In general, protocols for making RNA from eukaryotic organisms involve lysing the cells in the presence of a strong denaturant and deproteinizing agent which inhibits RNase as well as strips the protein away from the RNA. In this protocol, the RNA is then separated from DNA and other impurities by selective precipitation in high salt.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Diethylpyrocarbonate (DEPC)
  • Liquid nitrogen
  • recipeGrinding buffer
  • recipePhenol equilibrated with TLE solution
  • Chloroform
  • 8 M and 2 M LiCl
  • 3 M sodium acetate
  • 100% ethanol
  • Polytron ( Brinkmann PT 10/35)
  • Beckman JA‐10, JA‐20, and JA‐14 rotors (or equivalent)
  • 50‐ml Oak Ridge tube
  • Sarstedt tube
Sodium acetate, water, and LiCl solutions should be treated with DEPC to inhibit RNase activity. See unit 4.1, reagents and solutions, for instructions. CAUTION:DEPC is a suspected carcinogen and should be handled carefully.
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Figures

Videos

Literature Cited

Literature Cited
   Chirgwin, J.M., Przbyla, A.E., MacDonald, R.J., and Rutter, W.J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294.
   Kirby, K.S. 1968. Isolation of nucleic acids with phenolic solvents. Meth. Enzymol. 12B:87.
   Palmiter, R.D. 1974. Magnesium precipitation of ribonucleoprotein complexes: Expedient techniques for the isolation of undegraded polysomes and messenger ribonucleic acid. Biochemistry 13:3606.
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