MIT7_13f08_lab02_Protocol_Agarose - MIT OpenCourseWare...

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Unformatted text preview: MIT OpenCourseWare 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: . 7 . 1 3 F a l l 2 0 0 8 P a g e | 1 Agarose Gel Electrophoresis and DNA Band Excision How it works: Agarose gel electrophoresis is a simple and highly effective method for separating, identifying, and purifying 0.5 to 25 kb DNA fragments. Voltage applied at the ends of an agarose gel generates an electric field with a strength defined by the length of the gel and the potential difference at the ends (V/cm). DNA molecules exposed to this electric field migrate toward the anode (positive end) due to the negatively charged phosphates along the DNA backbone. The migration velocity is limited by the frictional force imposed by the gel matrix. While charge and/or size can affect the rate at which macromolecules will pass through the gel, the charge to mass ratio is the same for DNA molecules of different lengths. It is the size of the DNA, therefore, that determines the rate at which it passes through the gel, thereby allowing an effective separation of DNA fragment-length mixtures by electrophoresis. To visualize the DNA, the gel is treated with ethidium bromide. This dye intercalates between the stacked bases of nucleic acids and fluoresces bromide....
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MIT7_13f08_lab02_Protocol_Agarose - MIT OpenCourseWare...

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