Agarose Gel Electrophoresis

Daniel Voytas1

1 Iowa State University, Ames, Iowa
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.5A
DOI:  10.1002/0471142727.mb0205as51
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 protocol 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.

     
 
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Table of Contents

  • Section II: Resolution and Recovery of Large DNA Fragments
  • 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
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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

  Materials
  • Electrophoresis buffer ( TAE or TBE; appendix 22)
  • recipeEthidium bromide solution (see recipe)
  • Electrophoresis‐grade agarose
  • recipe10× loading buffer (see recipe)
  • DNA molecular weight markers (see Fig. )
  • 55°C water bath
  • Horizontal gel electrophoresis apparatus
  • Gel casting platform
  • Gel combs (slot formers)
  • DC power supply
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Figures

  •   FigureFigure 2.5.1 Minigel apparatus.
  •   FigureFigure 2.5.2 Effect of (A) agarose concentration, (B) applied voltage, (C) electrophoresis buffer, and (D) ethidium bromide on migration of DNA through agarose gels.
  •   FigureFigure 2.5.3 Migration pattern and fragment sizes of common DNA molecular weight markers.

Videos

Literature Cited

Literature Cited
   Griess, G.A., Louie, D., and Serwer, P. 1995. A desktop, low‐cost video fluorometer for quantitation of macromolecules after gel electrophoresis. Appl. Theor. Electrophor. 4:175‐177.
   Harriman, W.D. and Wabl, M. 1995. A video technique for the quantification of DNA in gels stained with ethidium bromide. Anal. Biochem. 228:336‐342.
   Helling, R.B., Goodman, H.M., and Boyer, H.W. 1974. Analysis of R. 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. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   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.
   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.
   Southern, E. 1979. Gel electrophoresis of restriction fragments. Meth. Enzymol. 68:152‐176.
   Sutherland, J.C., Lin, B., Monteleone, D.C., Mugavero, J., Sutherland, B.M., and Trunk, J. 1987. Electronic imaging system for direct and rapid quantitation of fluorescence from electrophoretic gels: Application to ethidium bromide‐ stained DNA. Anal. Biochem. 163:446‐457.
   Thorne, H.V. 1967. Electrophoretic characterization and fractionation of polyoma virus DNA. J. Mol. Biol. 24:203.
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