Agarose Gel Electrophoresis

Daniel Voytas1

1 Iowa State University, Ames, Iowa
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.4
DOI:  10.1002/0471142735.im1004s02
Online Posting Date:  May, 2001
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Abstract

Agarose gel electrophoresis is a simple and highly effective method for separating, identifying, and purifying 0.5‐ to 25‐kb DNA fragments. The Basic Protocol in this unit can be divided into three stages: (1) a gel is prepared with an agarose concentration appropriate for the size of DNA fragments to be separated; (2) the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation; and (3) the gel is stained or, if ethidium bromide has been incorporated into the gel and electrophoresis buffer, visualized directly upon illumination with UV light. Support protocols describe the use of midigels and minigels, as well as recommendations for photographing stained gels.

     
 
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Table of Contents

  • Basic Protocol 1: Resolution of DNA Fragments on Standard Agarose Gels
  • Support Protocol 1: Minigels and Midigels
  • Support Protocol 2: Photography of DNA in Agarose Gels
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Resolution of DNA Fragments on Standard Agarose Gels

  Materials
  • Electrophoresis buffer ( recipeTAEor recipeTBE)
  • recipeEthidium bromide solution
  • Electrophoresis‐grade agarose
  • recipe10× loading buffer
  • DNA molecular weight markers (Fig. )
  • Horizontal gel electrophoresis apparatus
  • Gel‐casting platform
  • Gel combs (slot formers)
  • DC power supply
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Figures

Videos

Literature Cited

Literature Cited
   Finney, M. 1988. Pulsed‐field gel electrophoresis. 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. 2.5.9‐2.5.15. Greene Publishing and Wiley‐Interscience, New York.
   Helling, R.B., Goodman, H.M., and Boyer, H.W. 1974. Analysis of EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose gel electrophoresis. J. Virol. 14:1235‐1244.
   Johnson, P.H. and Grossman, L.I. 1977. Electrophoresis of DNA in agarose gels. Optimizing separations of conformational isomers of double‐and single‐stranded DNAs. Biochemistry 16:4217‐4224.
   Maniatis, T., Fritsch, E.F., and Sambrook, J. 1989. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Sharp, P.A., Sugden, B., and Sambrook, J. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose. Biochemistry 12:3055‐3063.
   Thorne, H.V. 1967. Electrophoretic characterization and fractionation of polyoma virus DNA. J. Mol. Biol. 24:203.
Key References
   McDonell, M.W., Simon, M.N., and Studier, F.W. 1977. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol. 110:119‐146.
   Southern, E. 1979. Gel electrophoresis of restriction fragments. Methods Enzymol. 68:152‐176.
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