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Indiana University Bloomington

Electrophoresis

Gel electrophoresis is used to separate out DNA or RNA fragments according to their size using an electric current applied to a gel matrix. Samples are loaded in “wells” on the gel and at least one well is loaded with a “ladder” of fragments of known molecular weights. Voltage is applied to the gel and the contents of each well migrate down their respective “lane”. DNA or RNA fragments within each sample migrate across the gel at different rates according to their size. The differences in migration result in one or more bands within each lane of the gel, with each band representing a collection of identical fragments contained within the sample.

Agarose and polyacrylamide are the most common gel matrices used in DNA/RNA electrophoresis. After the electrophoresis procedure is complete, the different bands can be stained (with ethidum bromide or coomassie blue, usually) for easy visualization. Gel electrophoresis is often used to confirm DNA amplification of a specific sequence after PCR, or to confirm the presence of intact RNA following extraction. It can be used as the precursor to Northern or Southern blots. You also can remove DNA bands from the gel following electrophoresis, purify them and sequence them if desired.

Gel electrophoresis also can be used to separate proteins in a mixture. The protein electrophoresis method employs a gel matrix as before (usually a sodium dodecyl sulfate polyacrylamide gel; SDS-PAGE). Often times, it also incorporates a separation technique that uses isoelectric focusing (a pH gradient). This is called a two-dimensional or 2-D gel. Resultant bands (or spots, in the case of a 2-D gel) can be further characterized using Western blots or mass spectrophotometry.

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