05 - Biotechnology Basics: My First Electrophoresis
Introduction Agarose gel electrophoresis is a biotechnology technique used every day in research labs in labs worldwide. Electrophoresis uses electricity and a porous gel matrix to separate different molecules into discrete zones, or bands, based on physical properties like charge, size, and shape. In our dynamic workshop, we will dive into every step of the process—from loading and running gel, exploring the mechanisms of separation, and finally analyzing the results using two different electrophoresis systems. Background Information PRINCIPLES OF GEL ELECTROPHORESIS Agarose gel electrophoresis is a common laboratory technique used to separate molecules based upon charge, size and shape. It is particularly useful in separating charged biomolecules such as DNA, RNA and proteins. This technique possesses great resolving power, yet is relatively simple and straightforward to perform. Agarose, a polysaccharide derived from seaweed, is used to form the separation matrix used for gel electrophoresis. To make a gel, solid agarose powder is added to buffer and melted by boiling. The buffer controls the pH of the solution throughout the electrophoresis process, which influences the charge and stability of biological molecules. Once the solution has cooled to approximately 60° C, it is poured into a gel tray to solidify. A special comb is used to form depressions in the gel called loading wells. Once solidified, the gel is placed in a horizontal electrophoresis cham - ber and covered with a pH-balanced buffer. Electrodes placed at each end of the electrophoresis chamber generate current when connected to a direct current power supply. The buffer contains ions necessary to conduct the electrical current. Samples are prepared for electrophoresis by mixing them with glycerol or sucrose, which makes them denser than the electrophoresis buffer. When the samples are loaded into the wells, the dense samples sink through the buffer and remain in the wells. An electrical current is passed through the gel to drive molecules through the gel. Generally, the higher the applied voltage, the faster the samples are separated by electrophoresis. Once the current is applied, the following factors affect the mobil - ity of molecules through a gel: 1. Molecular size: On the molecular level, the gel contains small channels that act as a molecular sieve. Small molecules move through these holes easily, but larger ones have a more difficult time squeezing through the tunnels. 2. Gel concentration: The final concentration of agarose in a gel will change the size of the channels within the gel. Lower percent gels will have larger channels, making it easier to separate large molecules. Higher gel concentrations will have smaller channels, making it easier to separate small molecules. 3. Molecular charge: Given two molecules of similar size and shape, like dyes, the one with the greater amount of charge will migrate faster. Molecules with a net
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