Deprotection of Oligonucleotides and Purification Using Denaturing PAGE

Andrew Ellington1, Joanne Chory2

1 Indiana University, Bloomington, Indiana, 2 The Salk Institute, La Jolla, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.7
DOI:  10.1002/0471142735.im1007s06
Online Posting Date:  May, 2001
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Abstract

The advantages of purification by denaturing polyacrylamide gel electrophoresis (PAGE) are speed, simplicity, and high resolution. Although yields tend to be low (<50% of applied sample), the amount of material recovered is usually far in excess of that required for most molecular biology applications (e.g., cloning and sequencing). Denaturing PAGE can resolve oligonucleotides from 2 to 300 bases, depending on the percentage of polyacrylamide used. The method presented here is thus useful not only for isolating chemically synthesized deoxyribonucleotides but also small RNAs or other singleā€stranded polynucleotides.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Concentrated ammonium hydroxide
  • 10× and 1× TBE buffer, pH 8.0 (unit 10.4)
  • recipe40% acrylamide/2% bisacrylamide stock
  • TEMED (N,N,N′,N′‐tetramethylethylenediamine)
  • Urea
  • 10% ammonium persulfate
  • recipe2× formamide loading buffer
  • 0.3 M sodium acetate, pH 7.5
  • TE buffer (optional; appendix 2A)
  • Glass plates (20 × 16–cm), spacers (1.6‐mm), and combs for pouring gels
  • Acrylamide gel electrophoresis apparatus
  • Thin‐layer chromatography (TLC) plate with fluorescent indicator (e.g., Sigma #T7395)
  • Additional reagents and equipment for phenol extraction and ethanol precipitation (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
  Applied Biosystems, 1987. Evaluation and Purification of Synthetic Oligonucleotides. User Bulletin Issue 13 (see APPENDIX 5).
   Ellington, A. and Green, R. 1990. Synthesis of Oligonucleotides. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, eds.) pp. 2.11.1‐2.11.18. Greene Publishing and Wiley‐Interscience, New York.
   Frank, R., Müller, D., and Wollf, C. 1981. Identification and suppression of secondary structures formed from deoxyoligonucleotides during electrophoresis in denaturing polyacrylamide gels. Nucl. Acids Res. 9:4967‐4979.
   Maniatis, T., Jeffrey, A., and deSande, H.V. 1975. Chain length determination of small double‐and single‐stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14:3787‐3794.
   Smith, H.O. 1980. Recovery of DNA from gels. Methods Enzymol. 65:371‐379.
   Vorndam, A.V. and Kerschner, J. 1986. Purification of small oligonucleotides by polyacrylamide gel electrophoresis and transfer to diethylaminoethyl paper. Anal. Biochem. 152:221‐225.
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