Agarose gel electrophoresis

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Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.[1] Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.[2] Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins.

Contents

Applications

Agarose gels are easily cast and handled compared to other matrices and nucleic acids are not chemically altered during electrophoresis. Samples are also easily recovered. After the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator.

How to make 1% agarose gel: In a small beaker, mix 32mL of purified H2O, 4mL of 10x TBE buffer or 10x whichever buffer going to be used in electrophoresis, and 0.4g of agarose powder. Heat on hot plate, or microwave, until solution is almost boiling (about 98 degrees Celsius). Immediately pour solution into electrophoresis mold with notch (well) comb intact, let settle for approximately 30 minutes, or until it appears firm. Be careful, a gel which is perfectly molded is still extremely fragile. Remove notch (well) comb carefully, but quickly. Wrap in plastic and place in refrigerator if gel is not for immediate use.

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