Purification of Oligonucleotides Using Denaturing Polyacrylamide Gel Electrophoresis

Andrew Ellington1, Jack D. Pollard2

1 University of Texas, Austin, Texas, 2 Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.12
DOI:  10.1002/0471142727.mb0212s42
Online Posting Date:  May, 2001
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Abstract

Cloning vectors derived from filamentous phage are extremely useful because they allow cloned DNA to be isolated as either single‐ or double‐stranded DNA. This unit contains protocols for preparing both forms of DNA and for characterizing inserts in M13‐derived vectors. A protocol is also presented for preparing single‐stranded DNA from plasmids using superinfection with helper phage. This method is advantageous because it allows cloned DNA to be maintained in the form of a plasmid while permitting single‐stranded DNA to be isolated for DNA sequencing.

     
 
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Table of Contents

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

Basic Protocol 1:

  Materials
  • Nucleic acid samples
  • Urea
  • 10× and 1× TBE buffer, pH 8 ( appendix 22)
  • 40% acrylamide/2% bisacrylamide (unit 7.6)
  • TEMED (N,N,N′,N′‐tetramethylethylenediamine)
  • 10% ammonium persulfate (APS) in water (store ≤1 month at 4°C)
  • recipeUrea loading buffer (see recipe)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • TE buffer, pH 7.5 ( appendix 22)
  • Acrylamide gel electrophoresis apparatus
  • Glass plates, spacers, and combs for pouring gels
  • 50°, 60° and 90°C water baths
  • DC power supply
  • Thin‐layer chromatography (TLC) plate with fluorescent indicator (e.g., Silica Gel F‐254 or IB‐F, Merck)
  • Hand‐held short‐wave (254‐nm) UV lamp
  • Small‐bore (5‐ml) syringe (e.g., Becton Dickinson)
  • 15‐ml centrifuge tube capable of withstanding high temperatures
  • Rotary shaker
  • 0.2‐µm filter (Gelman Sciences)
  • Additional reagents and equipment for oligonucleotide deprotection (unit 2.11), gel electrophoresis (unit 2.7), phenol extraction, and ethanol and butanol precipitation (unit 2.1)
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Figures

Videos

Literature Cited

Literature Cited
  Applied Biosystems. 1984. Evaluation and Purification of Synthetic Oligonucleotides. User Bulletin no. 13. Applied Biosystems Foster City, Calif.
   Chen, Z. and Ruffner, D.E. 1996. Modified crush‐and‐soak method for recovering oligodeoxynucleotides from polyacrylamide gel. Biotechniques 21:820‐822.
   Frank, R., Muller, D., and Wolff, 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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