Biology 2960 Computer Laboratory

DNA Gel Electrophoresis


"THE BIG PICURE"

DNA may be analyzed by gel electrophoresis. In this process, DNA molecules migrate to the "+" pole as an electrical current passes through the gel. DNA molecules of different lengths separate with the shorter fragments moving faster through the gel compared to the larger fragments. In addition, the "shape" (linear or circular) of the DNA molecule will affect the migration of the DNA molecule.

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Step 1: Making the gel - A common material used for gel electrophoresis of DNA is agarose. Agarose gels are made by first boiling a mixture of powdered agarose and buffer. When the mixture is cooled to about 65 º C, the solution is poured into a gel mold. When further cooled to room temperature, the agarose solidifies to produce the gel with indentations called wells. (Note that in our lab gels, ethidium bromide (EtBr) was added immediately prior to pouring the gel. EtBr allows us to see the DNA with UV light exposure).

Step 2: Setting up the electrophoresis apparatus - The agarose gel is placed in an electrophoresis apparatus and buffer is added to cover the gel.


Step 3: Loading the gel - The agarose gel is placed in an electrophoresis apparatus and buffer is added to cover the gel.

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Step 4: Movement of the DNA fragments - After the wells are filled, the power supply is turned on and the DNA moves toward the "+" (positive) pole. At this stage the DNA band(s) cannot be seen, but the tracking dye (dark blue) allows the progress of the electrophoresis to be followed. The smallest DNA fragments typically follow the blue dye down the gel.

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Step 5: Staining the DNA bands - The DNA is visualized by binding to ethidium bromide (EtBr) as it migrates through the gel. Recall that EtBr was added to the agarose gel prior to cooling (solidifying). The EtBr intercalates (fits between the base pair stacks) into the DNA and makes it fluoresce orange when exposed to UV light. A photograph/image of the gel is often taken to have a permanent record of the experiment and for analysis.

Step 6: Analysis of DNA Fragments - Often, it is important to determine the sizes of DNA bands on gels after electrophoresis. This may easily be done if a marker DNA sample is run alongside the experimental samples on the gel. The marker DNA sample contains a number of DNA molecules, each with a known molecular size. For example, bacteriophage lambda genomic DNA digested with HindIII has 8 fragments of 23130, 9416, 6557, 4361, 2322, 2027, 564, and 125 base pairs. (The 125 bp fragment is often not seen in experiments, either because it is too faint or because it runs off the gel.)




Adapted from Pearson Education, Inc. publishing as Pearson Prentice Hall © 2007 All Rights Reserved