Agarose Gel Electrophoresis

Jennifer A. Armstrong1, Joseph R. Schulz2

1 Joint 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.et0702s00
Online Posting Date:  October, 2008
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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.

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 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
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: DNA Agarose Gel Electrophoresis

  • 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
  • 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

  • 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)
  • Formamide
  • Formaldehyde loading buffer (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
  • Horizontal gel electrophoresis system, including a buffer chamber, gel tray, and comb
  • Electrophoresis power supply
  • 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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  •   FigureFigure 7.2.1 A negatively charged phosphate group connects nucleosides through phosphodiester linkages in nucleic acid polymers.
  •   FigureFigure 7.2.2 Chemical structure of the repeating D‐galactose, 3,6‐anhydro L‐galactose disaccharide of agarose. Hydrogens bound to carbons are omitted for clarity.
  •   FigureFigure 7.2.3 (A) Planar structure of ethidium bromide and (B) stick‐model representation of ethidium bromide intercalated into a C,G base‐pair stack. Hydrogen bonding of the base pairs is indicated by gray lines. The bromide ion is the counterion for the positively charged ethidium molecule; the nitrogen of ethidium bromide has a positive charge. In the electric field, the positively charged ethidium will migrate toward the negative pole, and it is that molecule which is seen under UV light.
  •   FigureFigure 7.2.4 Pouring the agarose gel. (A) Addition of agarose to 1× TAE running buffer. (B) After dissolving the agarose in a microwave, the gel solution is clear, with no transparent specks of agarose evident. (C) Once the gel solution has cooled to allow handling (55° to 60°C), it can be poured. For the gel rig pictured, the gel tray is placed in the buffer chamber 90° with respect to the usual running orientation, and the gel is poured. Rubber gaskets in the sides of the gel tray prevent leaking. Note the presence of the comb (arrow). This particular comb is double sided, with one set of teeth thicker than the other to allow a choice of narrow or thick wells.
  •   FigureFigure 7.2.5 Loading the agarose gel. (A) The gel has fully set when it looks translucent, but not clear in any spots; this usually takes ∼30 min. Pull the comb (arrow) straight up in a slow, smooth motion, using two hands. In the gel rig pictured, the tray will be lifted out, rotated 90°, and replaced in the buffer chamber. (B) When loading the gel, stabilize the micropipettor with your other hand. Avoid tearing or puncturing the well with the pipet tip.
  •   FigureFigure 7.2.6 Running the agarose gel. (A) Place the lid on the buffer chamber and connect the leads to the power supply. (B) Orient the lid such that the black power supply lead (the cathode, negative lead) is behind the DNA lanes, with the DNA running towards the red power supply lead (the anode, positive lead). This gel rig has the leads built into the cover.
  •   FigureFigure 7.2.7 Linear DNA migrates according to its size. Shown are two 0.8% agarose gels loaded with identical DNA samples and run at 100 V until the bromphenol blue was three‐quarters down the gel (∼50 min). 500 ng of 1 Kb Plus DNA ladder (Invitrogen) was loaded into lanes 1 and 5; 300 ng of DNA was loaded into lanes 2 to 4 and 6 to 8. Lanes 2 and 6 contain the linear plasmid (6.4 kb); lanes 3 and 7 contain two DNA fragments, the vector (5.7 kb), and the DNA insert (738 bp); lanes 4 and 8 contain the uncut plasmid (supercoiled). Note that, although the supercoiled plasmid is 6.4 kb, it migrates significantly further than 6.4‐kb linear DNA (for example compare lane 4 to lane 2). The slower‐migrating band in lanes 4 and 8 is relaxed (nicked) circular plasmid. The gel on the left was stained with ethidium bromide after the gel run was complete. Ethidium bromide (EtBr) was added to the gel on the right, allowing the DNA to be stained during the run. Note the insert DNA band of 738 bp (white arrows) is more difficult to detect in the gel on the right. The gels are shown at actual size.
  •   FigureFigure 7.2.8 Standard curves of DNA size standards in Figure . Note that inclusion of ethidium bromide (EtBr) in the gel results in a reduction in the distance migrated by DNA. This is evidenced by the shift to the left of the standard curve. The standard curve can be used to estimate the size of other DNA bands on the same gel. The dashed line at 5.7 cm corresponds to the insert DNA (white arrow in Figure ).


Literature Cited

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