Separation of Small DNA Fragments by Conventional Gel Electrophoresis

Joanne Chory1, Jack D. Pollard2

1 The Salk Institute for Biological Studies, La Jolla, California, 2 Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.7
DOI:  10.1002/0471142727.mb0207s47
Online Posting Date:  May, 2001
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Abstract

Large amounts of small (<1000‐bp) DNA fragments can be separated by conventional electrophoretic means. The purified fragments can then be used for cloning, sequencing, and labeling. In this unit, the techniques of DNA separation via both nondenaturing polyacrylamide and sieving agarose electrophoresis are discussed. Methods are detailed for the pouring and electrophoresis of nondenaturing polyacrylamide gels, followed by elution of the labeled or unlabeled separated DNA fragments from the gels by either passive diffusion or electroelution. A method is also provided for the use of sieving agarose, a specially treated type of agarose designed to be used at high concentrations. Poured and run like conventional agarose gels, this matrix can resolve small DNA fragments much like a nondenaturing polyacrylamide gel.

     
 
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Table of Contents

  • Section III: Resolution and Recovery of Small DNA Fragments
  • Basic Protocol 1: Nondenaturing Polyacrylamide Gel Electrophoresis
  • Alternate Protocol 1: Electroelution of Small DNA Fragments from Polyacrylamide Gels
  • Basic Protocol 2: Sieving Agarose Gel Electrophoresis
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Nondenaturing Polyacrylamide Gel Electrophoresis

  Materials
  • 10× and 1× TBE electrophoresis buffer, pH 8.0 ( appendix 22)
  • recipe29:1 (w/w) acrylamide/bisacrylamide (see recipe; solutions are also available commercially from National Diagnostics)
  • TEMED (N,N,N′,N′‐tetramethylethylenediamine; store at 4°C)
  • 10% (w/v) ammonium persulfate (APS) in water (store ≤1 month at 4°C)
  • recipe5× loading buffer (see recipe)
  • DNA samples
  • DNA‐molecular‐weight markers: e.g., pBR322 restriction digested with HinfI or M13 digested with HpaII
  • 0.5 µg/ml ethidium bromide
  • recipeElution buffer, pH 7.5 (see recipe)
  • 100% and 70% ethanol
  • TE buffer, pH 7.5 ( appendix 22)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • Glass plates, spacers, and combs for casting gels
  • Acrylamide gel electrophoresis apparatus
  • DC power supply
  • Thin‐layer chromatography (TLC) plate with fluorescent indicator (e.g., Silica Gel F‐254 or IB‐F; for UV shadowing)
  • Longwave UV transilluminator
  • 3‐ml small‐bore disposable syringe
  • Syringe equipped with silanized glass wool plug (unit 5.6) or 2‐µm filter
  • Centrifuge with Beckman JA‐20 rotor or equivalent
  • Additional reagents and equipment for ethanol precipitation (unit 2.1)

Alternate Protocol 1: Electroelution of Small DNA Fragments from Polyacrylamide Gels

  • 0.5× TBE electrophoresis buffer, pH 8.0 ( appendix 22)
  • Small dialysis bag
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)

Basic Protocol 2: Sieving Agarose Gel Electrophoresis

  Materials
  • Sieving agarose (NuSieve GTG agarose)
  • TAE or TBE electrophoresis buffer, pH 8.0 ( appendix 22)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and isolating DNA using low gelling/melting temperature agarose (unit 2.6)
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Figures

Videos

Literature Cited

Literature Cited
   Maniatis, T. and Ptashne, M. 1973a. Multiple repressor binding at the operators in bacteriophage. Proc. Natl. Acad. Sci. U.S.A. 70:1531‐1535.
   Maniatis, T. and Ptashne, M. 1973b. Structure of the operators. Nature 246:133‐136.
   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.
Key Reference
   Chrambach, A. and Rodbard, D. 1971. Polyacrylamide gel electrophoresis. Science 172:440‐451.
  Provides background chemistry of polymerization of acrylamide and reviews the size separation characteristics of gels with different acrylamide percentages.
   FMC Marine Colloids product information.
  Sieving agarose was developed by FMC Marine Colloids. Literature describing its properties and use is available from the company.
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