Polyacrylamide Gel Electrophoresis (PAGE) of Synthetic Nucleic Acids

Alex Andrus1, Robert G. Kuimelis2

1 PE Applied Biosystems, Foster City, California, 2 Phylos, Inc., Lexington, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.4
DOI:  10.1002/0471142700.nc1004s01
Online Posting Date:  May, 2001
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Abstract

Protocols are given for analysis of oligonucleotides by PAGE, using either methylene blue staining or radiolabeling to mark the oligonucleotide. In addition, a separate protocol is provided for purification by PAGE.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Synthetic Nucleic Acids by PAGE
  • Support Protocol 1: Visualization Using Methylene Blue Staining
  • Support Protocol 2: Radiolabeling of Oligonucleotides Using T4 Polynucleotide Kinase
  • Basic Protocol 2: Purification of Synthetic Nucleic Acids Using Page and UV Shadowing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Synthetic Nucleic Acids by PAGE

  Materials
  • 38% (w/v) acrylamide/2% (w/v) bisacrylamide (see recipe)
  • 1× formamide/dye mix (see recipe for 10×; dilute in TBE buffer)
  • Loading buffer: 9:1 formamide/ 1× TBE buffer
  • Vertical electrophoresis system, such as Hoefer Model SE 400 or SE 600 in the 16 × 18–cm format with power supply (Pharmacia Biotech)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis ( appendix 3B)

Support Protocol 1: Visualization Using Methylene Blue Staining

  • 0.02% (w/v) methylene blue (Aldrich) in H 2O

Support Protocol 2: Radiolabeling of Oligonucleotides Using T4 Polynucleotide Kinase

  Materials
  • Radiolabeling master mix (see recipe)
  • 10× formamide/dye mix (see recipe)
  • Additional reagents and equipment for preparing and running a polyacrylamide gel ( protocol 1)

Basic Protocol 2: Purification of Synthetic Nucleic Acids Using Page and UV Shadowing

  • 20 × 20–cm fluorescent TLC plate, wrapped with UV‐transparent plastic wrap
  • Hand‐held, short‐wavelength UV light (240 to 300 nm)
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Figures

Videos

Literature Cited

Literature Cited
   Andrus, A. 1992. Evaluating and Isolating Synthetic Oligodeoxynucleotides. PE Biosystems Division of Perkin‐Elmer, Foster City, Calif. Available upon request.
   Efcavitch, J.W. 1990. The electrophoresis of synthetic oligonucleotides. In Gel Electrophoresis of Nucleic Acids—A Practical Approach (D. Rickwood and B.D. Hames, eds.) pp. 125‐149. Oxford University Press, Oxford.
   Ellington, A. and Pollard, J.D. Jr. 1998. Purification of oligonucleotides using denaturing polyacrylamide gel electrophoresis. In Current Protocols in Molecular Biology, vol. 1 (F. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.12.1‐2.12.7. John Wiley & Sons, New York.
   Maniatis, T., Jeffrey, A., and van deSande, H. 1975. Chain length determination of small double‐ and single‐stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14:3787‐3794.
   Molecular Probes 1996. Handbook of Fluorescent Probes and Research Chemicals, 6th ed. pp. 164.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Wallace, R.B. and Miyada, C.G. 1987. Oligonucleotide probes for the screening of recombinant DNA libraries. In Methods in Enzymology, Vol.152, Guide to Molecular Cloning Techniques (S.L. Berger and A.R. Kimmel, eds.) pp. 432‐442. Academic Press, San Diego.
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