Agarose Gel Electrophoresis

Jennifer A. Armstrong1, Joseph R. Schulz2

1 W.M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, California, 2 Occidental College, Los Angeles, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 7.2
DOI:  10.1002/9780470089941.et0702s10
Online Posting Date:  May, 2015
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Abstract

Agarose gel electrophoresis, which separates and sizes linear DNA and RNA fragments, is arguably the most basic and essential technique in molecular biology. It is commonly employed for analysis of PCR products, plasmid DNA, and products of restriction enzyme digestion. It is the first step for analysis of specific DNA and RNA fragments by northern and Southern blots. In this unit, we provide both written instructions and photographic images to take the reader from preparing a first agarose gel to analyzing results and determining the size of sample DNA. We include two protocols: agarose gel electrophoresis (commonly used to analyze DNA) and denaturing gel electrophoresis (for analyzing RNA). We have divided each protocol into four basic steps: (1) preparing and pouring the agarose gel; (2) preparing and loading samples; (3) running the agarose gel; and (4) staining the gel using the fluorescent stain ethidium bromide to visualize DNA and RNA. © 2015 by John Wiley & Sons, Inc.

Keywords: DNA; RNA; separation; sizing; ethidium bromide; voltage; TAE; TBE; bromphenol blue; xylene cyanol; northern blotting; Southern blotting

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Questions
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: DNA Agarose Gel Electrophoresis
  • Basic Protocol 2: Denaturing RNA Agarose Gel Electrophoresis
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Variations
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: DNA Agarose Gel Electrophoresis

  Materials
  • Running buffer: 1× TAE running buffer (see recipe for 50×) or 0.5× TBE running buffer (see recipe for 5×)
  • Agarose, molecular‐biology grade
  • 6× loading buffer (see recipe)
  • DNA samples to be run
  • DNA size standard (often called DNA ladder or DNA marker)
  • Ethidium bromide working stain solution (see recipe)
  • Erlenmeyer flask of appropriate size for preparation of agarose gel
  • Autoclave gloves
  • Horizontal gel electrophoresis system, including a buffer chamber, gel tray, comb
  • Electrophoresis power supply and electrical leads
  • Containers for staining and destaining gels (Rubbermaid‐type work well)
  • Platform shaker or orbital shaker
  • UV light box
  • Gel documentation system: Polaroid film–based or CCD digital–camera based

Basic Protocol 2: Denaturing RNA Agarose Gel Electrophoresis

  Materials
  • Agarose, molecular biology grade
  • 10× and 1× MOPS running buffer (see recipe)
  • 37% (12.3 M) formaldehyde, pH >4.0
  • RNase‐free H 2O (unit 8.2; Hoopes, )
  • Formamide
  • RNA loading dye (see recipe)
  • RNA molecular weight ladder and/or RNA of known size
  • 0.5 M ammonium acetate
  • 0.5 μg/ml ethidium bromide in 0.5 M ammonium acetate (prepare from 10 mg/ml ethidium bromide stock)
  • Erlenmeyer flask of appropriate size for preparation of agarose gel
  • Autoclave gloves
  • 55°C water bath or heat block
  • RNase‐free horizontal gel electrophoresis system, including a buffer chamber, gel tray, and comb
  • Electrophoresis power supply and electrical leads
  • RNase‐free containers for staining and destaining gels (Rubbermaid‐type work well; see unit 8.2 for elimination of RNase contamination)
  • Platform or orbital shaker
  • UV box
  • Gel documentation system: Polaroid‐film based or CCD digital digital–camera based
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Figures

Videos

Literature Cited

Literature Cited
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Internet Resources
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