5th Lecture Agarose gel electrophoresis (1).pdf

AGAROSE GEL ELECTROPHORESIS Dr N.M.C. Nayanakantha Senior Lecturer (Grade I) Dept. of Biosystems Technology Faculty of Technological Studies What is electrophoresis? Electrophoresis is a technique that uses electrical current to separate DNA, RNA or proteins based on...

AGAROSE GEL ELECTROPHORESIS Dr N.M.C. Nayanakantha Senior Lecturer (Grade I) Dept. of Biosystems Technology Faculty of Technological Studies What is electrophoresis? Electrophoresis is a technique that uses electrical current to separate DNA, RNA or proteins based on their physical properties such as size and charge. DNA RNA Protein What is agarose gel electrophoresis? Agarose gel electrophoresis is a form of electrophoresis used for the separation of nucleic acid (DNA or RNA) fragments based on their size. Negatively charged DNA/RNA migrates through the pores of an agarose gel towards the positively charged end of the gel when an electrical current is applied. Smaller fragments migrating faster than larger fragments. The resulting bands can then be visualized using ultraviolet (UV) light. Agarose gel As DNA is not visible to the naked eye, an intercalating dye such as Ethidium Bromide (EtBr) is incorporated in the gel during setting. This binds the DNA and fluoresces under UV light, allowing the DNA fragments to be visualized. The more DNA present, the brighter the band. Equipment for Gel Electrophoresis Protocol for gel preparation and running buffer  Composition of DNA Loading Buffer It is a solution of 30mM EDTA, 36% (v/v) Glycerol, 0.05% (w/v) Xylene Cyanol FF, 0.05% (w/v) Bromophenol Blue for use in agarose gel electrophoresis of nucleic acids. Visualizing a gel using a Gel Doc Equipment