Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Most every molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and analysis of DNA. We will be using agarose gel electrophoresis to determine the presence and size of PCR products.
Electrophoresis is a method of separating substances based on the rate of movement while under the influence of an electric field. Agarose is a polysaccharide purified from seaweed. An agarose gel is created by suspending dry agarose in a buffer solution, boiling until the solution becomes clear, and then pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-like slab. During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a positive and negative electrode. The DNA to be analyzed is forced through the pores of the gel by the electrical current. Under an electrical field, DNA will move to the positive electrode (red) and away from the negative electrode (black). Several factors influence how fast the DNA moves, including; the strength of the electrical field, the concentration of agarose in the gel and most importantly, the size of the DNA molecules. Smaller DNA molecules move through the agarose faster than larger molecules. DNA itself is not visible within an agarose gel. The DNA will be visualized by the use of a dye that binds to DNA.
Purpose: To determine the presence or absence of PCR products and quantify the size (length of the DNA molecule) of the product.
Materials needed: Agarose TAE Buffer 6X Sample Loading Buffer DNA ladder standard Electrophoresis chamber Power supply Gel casting tray and combs DNA stain Staining tray Gloves Pipette and tips
Recipes: TAE Buffer 4.84 g Tris Base 1.14 ml Glacial Acetic Acid 2 ml 0.5M EDTA (pH 8.0) - bring