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Electrophoresis_Set_Up



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Electrophoresis The purpose of electrophoresis is to separate molecules of DNA RNA or protein Separation can be based upon 1 Size 2 Shape 3 Isoelectric point protein only Separation is achieved by using a matrix gel and an electromotive force to propel the molecules through the gel 01 14 19 1 Setting Up an Electrophoresis Gel To run a gel electrophoresis you will need 01 14 19 Gel tray Gel comb Electrophoretic gel box Agarose powder Buffer solution Dye Electric field 2 Electrophoresis involves 1 Preparation of the gel tray 2 Melting the agarose in a buffer solution 3 Addition of dye to the molten agarose solution optional 4 Pouring the molten agarose into the casting tray with a comb 5 Letting the gel solidify 6 Submerging the solid gel in buffer solution in the gel box 7 Removing the comb to reveal the wells in the gel 8 Loading the samples into the wells 9 Applying the electric current and running the gel 10 Analyzing the samples separated in the gel 01 14 19 3 Determining the size of gel needed One must first decide what size gel will be run In general For a small gel tray about 50 ml of gel is needed For a medium gel tray about 100 ml of gel is needed For a large gel tray about 200 ml of gel is needed note these are estimates the volumes will vary with the apparatus used 01 14 19 small medium large 4 Preparing Your Gel Tray To form a mold for the hot liquid gel the tray must be taped on the open sides To accomplish this pull one piece of masking tape over the open side leaving about 1 cm on the bottom to fold it over thus making a good seal Pour the gel in and insert the gel comb Let the gel cool Remove the tape and the comb and the gel is ready for use 01 14 19 Note some electrophoresis apparatuses come with gel casting boxes 5 Making the Gel Solution Weigh out the appropriate amount of agarose powder For example 100ml of a 1 6 Agarose solution will need 1 6 grams of agarose Pour the agarose powder into a Erlenmeyer flask the flask should be at least 2X the



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