Agarose Gel Electrophoresis for the Separation of DNA Fragments
Pei Yun Lee, John Costumbrado, Chih-Yuan Hsu, Yong Hoon Kim
Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles
Video Article Chapters
1:31 Preparation of the Gel
3:21 Setting up Gel Apparatus and Separating DNA Fragments
4:55 Observing Separated DNA Fragments
5:43 Results: Agarose Gel Electrophoresis of PCR Products
Cite this Article
Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. Agarose Gel Electrophoresis for the Separation of DNA Fragments. J. Vis. Exp. (62), e3923, doi:10.3791/3923 (2012).
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The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to:
Understand the mechanism by which DNA fragments are separated within a gel matrix
Understand how conformation of the DNA molecule will determine its mobility through a gel matrix
Identify an agarose solution of appropriate concentration for their needs
Prepare an agarose gel for electrophoresis of DNA samples
Set up the gel electrophoresis apparatus and power supply
Select an appropriate voltage for the separation of DNA fragments
Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands
Determine the sizes of separated DNA fragments
1. Preparation of the Gel
Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared using a w/v percentage solution. The concentration of agarose in a gel will depend on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The volume of the buffer should not be greater than 1/3 of the capacity of the flask.
Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA).
Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave, but can also be done over a Bunsen flame. At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved.
Add ethidium bromide (EtBr) to a concentration of 0.5 μg/ml. Alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-30 min, followed by destaining in running buffer for an equal length of time.
Note: EtBr is a suspected carcinogen and must be properly disposed of per institution regulations. Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the staining of DNA are available; however EtBr remains the most popular one due to its sensitivity and cost.
Allow the agarose to cool either on the benchtop or by incubation in a 65 °C water bath. Failure to do so will warp the gel tray.
Place the gel tray into the casting apparatus. Alternatively, one may also tape the open edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create the wells.
Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box. Alternatively, the gel can also be wrapped in plastic wrap and stored at 4 °C until use (Fig. 1).
2. Setting up of Gel Apparatus and...