Separating Restriction Fragments of Genomic DNA by Horizontal Agarose Gel Electrophoresis
Electrophoresis is a separation method that functions because charged particles or molecules migrate through a solution under the influence of an electric field. DNA fragments are charged molecules and bear a net negative charge because of the phosphate backbone. Therefore, DNA fragments will migrate towards the positive electrode (anode). The viscosity provided by the agarose allows the DNA to separate according to the molecular size and conformation during electrophoresis. DNA fragments ranging in size from 0.5–30 kilobases (kb) can be separated using agarose electrophoresis. Agarose gels are widely used in the electrophoretic separation of DNA fragments. Agarose is a highly purified and uncharged polysaccharide derived from agar. It dissolves in boiling water and remains liquid at temperatures over 40°C and becomes a stable gel at lower temperatures. The concentration of the agarose can be varied to obtain the suitable pore size. The higher the agarose concentration, the smaller the pore size. An agarose concentration of 0.6–0.7% is generally used in the electrophoretic analysis of DNA. In this experiment, the smaller DNA fragments resulting from the restriction endonuclease digestion of genomic DNA of rhizobia are separated by horizontal gel electrophoresis. The DNA fragments are stained with ethidium bromide (EtBr) and the banding patterns of the rhizobia are visualized by 302 nm UV illumination.
KeywordsPhosphate Backbone Bromphenol Blue Agarose Concentration Casting Unit Suitable Pore Size
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