Agarose Gel Electrophoresis of DNA Agarose gel electrophoresis is used to analyze the quality of DNA samples.

With the use of appropriate size markers, it is used to assess the integrity of the DNA and to calculate the sizes and concentrations of the various DNA fragments. The agarose must be molecular biology grade. The most commonly used gels are 1% gels, because this pore size allows good resolution of sizes from about 200 bp to 10 kb. The concentration can be increased or decreased to allow better resolution of low or high molecular weight species. Either TAE (Tris-acetate-EDTA) or 0.5 x TBE (Tris-borate-EDTA) can be used as the buffer system. TBE provides better buffering and gives better separation of low molecular weight species, but it can reduce the yield when gels are being used to isolate specific DNA fragments. See the bottom of this document for the composition of the various solutions used for DNA electrophoresis. 1. Prepare the molten agarose solution. Determine the appropriate volume of gel solution for the apparatus that you are using. For a 1% (1 g/ 100 mL) gel, weigh out the appropriate amount of agarose and place it in an Erlenmeyer flask with the appropriate volume of TAE, 0.5% TBE, or SYBR Safe. SYBR Safe (Invitrogen Corp.) solution contains SYBR Green fluorescent dye dissolved in 0.5 x TBE.) TAE and 0.5 x TBE buffers are diluted from concentrated stocks, while SYBR Safe is used directly. Microwave the flask until the agarose is dissolved. The best gels are made from agarose that has NOT been overcooked. Therefore, it is a good idea to do this in bursts of about 15-20 sec. After each burst, remove the flask and swirl it a bit (use a paper towel to protect your hand from the heat.) Stop when you see that the agarose has dissolved. 2. Pour the gel. Allow the agarose solution to cool, with occasionally swirling, until the flask is "very warm" to the touch. Pour the gel. Place the sample comb in place. Let the gel sit for at least 15 min before loading your samples. Before loading the gel, gently pour either TAE or 0.5 x TBE running buffer over it (These are called submarine gels.). This is a good stopping place gels can sit for several hours before loading. 3. Prepare your samples for loading on the gel. Before loading, each DNA sample needs to be mixed with 6X sample buffer, i.e. 1 vol sample buffer for 5 vol sample. (The sample buffer includes bromophenol blue and xylene cylanol tracking dyes that can be used to track the migration of samples as well as glycerol, which makes the samples dense enough to sink into the wells. Bromophenol blue is a dark blue dye that migrates like a 300 bp DNA fragment. Xylene cyanol is a turquoise dye that migrates like a 4 kb DNA fragment.) Convenient sample volumes are 5-15 l, depending on which comb you use. Each gel should include at least one lane with a DNA size marker. Size markers are sold by a variety of vendors. Details of the markers can be found in vendors' catalogs. We use several size markers. Standard III is a Eco RI/Hind III digest of phage. Because the fragments are present in equimolar amounts, it is possible to use this standard to estimate the quantity of DNA in an unknown sample. The standard is diluted so that 5 l contains 500 ng DNA.

Standard X is a DNA ladder constructed of artificially generated fragments that differ in size from one another in approx. 1 kb increments. This breaks down below 1 kb. Standard X gives much more resolution below 1 kb than does Standard III and should be used when you are analyzing small pieces of DNA. Note: The fragments are NOT represented in equimolar amounts, so this standard cannot be used to estimate DNA concentrations.

4. Run the gel. Connect the gel to the power source, matching the red (positive) and black (negative) connections. DNA is a negatively charged molecule and will be attracted to the positive (red) pole. Gels should be run at a constant voltage of 80-120 volts. Run the gel until the bromphenol blue tracking dye is about 1 cm from the end of the gel. Turn off the power before disconnecting the gel. 5. Stain the gel and visualize DNA. DNA fragments are visualized by staining gels with ethidium bromide (EtBr), an intercalating agent. Transfer the gel to a small tray containing distilled water. Add EtBr from a concentrated stock (stocks are stored refrigerated in the dark) to a final concentration of 0.5 g/ml. Rock the tray gently for approx. 30 min. (Gels made with SYBR Safe can be viewed without further staining.) Transfer the gel with a spatula to the transilluminator, turn on the UV light and photograph the gel. Turn off the UV light immediately after photographing the gel. SAFETY CONSIDERATIONS: Wear disposable gloves and eye protection for this step. EtBr is a potential mutagen, so be sure to handle the gels with gloves. Ultraviolet illumination is used to visualize DNA fragments, so eye protection is required.

50X TAE: 40 mM Tris-acetate, 1 mM EDTA To make l L: Combine 242 g Tris buffer, 57.1 ml acetic acid, 100 ml 0.5 M EDTA (pH 8.0) Revised 2/17/08 COC