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Gel electrophoresis is a technique used in molecular biology…

Gel electrophoresis is a technique used in molecular biology to separate a variety of types of polar molecules by applying electricity using a gel matrix. This method sorts of molecules by size, due to being run through a gel matrix containing pores and separates molecules by charge due to the applied electrical field. The gel matrix is made of agarose, a substance extracted from seaweed. Agarose is neutral in charge; thus, it does not interfere with the separation of sample molecules during gel electrophoresis. When agarose is mixed with an aqueous solution, it makes a semisolid gel medium, which is porous. Much like a sponge, sample molecules in the gel will move about according to their sizes and how they are restricted by the size of the pores. The more concentrated the gel mixture, the smaller the pores will be, which will cause molecules to move slower as they migrate through the gel. To get the molecules to move through these agarose pores, gel electrophoresis requires the application of an electrical field. Positively charged molecules will be attracted to the negatively charged electrode (cathode) of the gel chamber. Negatively charged molecules (such as DNA and RNA) will be attracted to the positively charged electrode (anode) of the gel apparatus. Also, as molecules travel on the gel, smaller molecules travel faster than larger molecules. Over time, the mixture of molecules separates into bands. All of this is accomplished in a gel electrophoresis chamber. This technique works well for separating DNA and RNA because these macromolecules are negatively charged. The phosphate molecules on the backbones of DNA and RNA are highly negative. Therefore, when DNA or RNA samples are loaded into the wells of the agarose gel, they move toward the anode of the electrophoresis chamber. A gel is placed in a buffered solution (TAE buffer) to apply to the electrical field. In an electrical field, electrons (electricity) are able to move more quickly with less resistance. The buffer serves to provide the necessary ions to conduct electricity, but also serves to maintain a stable pH and temperature during gel electrophoresis. When an electrical field is run through an aqueous solution, naturally, water molecules ionize as the molecular movement increases. 2H2O   ->  H3O+ + OH- The increased H+ in solution results in a change of pH and an increase in temperature. To keep the gel from melting during the electrophoresis process, it is essential to use a buffer in the chamber. Loading samples into an agarose gel Use a clean micropipette tip between each sample. Steady hand and slow release of the sample solution into the well. Results Molecules of difference size and charge will separate. Smaller molecules will migrate faster than larger molecules. Positively charged molecules will migrate toward cathode (the negative electrode). Negatively charged molecules will migrate toward anode (the positive electrode). The “thicker” the band, the more molecules are in that spot.