Cloning and Mapping

DNA Analysis

Molecular biology and, specifically, recombinant DNA technologies require rapid and accurate discrimination of DNA fragment size. Agarose or polyacrylamide gel-electrophoresis is the standard method used for separation, identification and purification of DNA fragments. DNA is visualized on a gel after soaking or pre-impregnating the gel with ethidium bromide, a DNA intercalating agent that fluoresces under UV illumination. DNA markers and ladders are essential for measuring DNA fragment size of digested DNA to ensure the desired DNA fragments are investigated. The original DNA markers were made of genomic DNAs digested with a restriction enzyme to exhibit a banding pattern of known fragment sizes. Later, markers were made of fragments with evenly-spaced sizes and the resulting banding pattern resembles a ladder. Using the marker or ladder as a reference, it is possible to determine the size and relative quantity of the DNA of interest. The bands are visible under UV illumination; they are not visible under normal lighting conditions. To track the progress of the gel as it runs, the marker contains a dye or combination of dyes that identify the leading edge of well contents, also called the dye front.

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Protocols for DNA Analysis