Gel Electrophoresis - Welcome to the West High School Site 凝胶电泳-欢迎来到西中学网站.pptVIP

A tool for separating specific biomolecules from a mixture(A key step in most biotechnology applications) Electrophoresis: movement of charged particles through a substance when an electric field is applied. The Gel Substance Agarose (from seaweed) Polyacrylamide Powders that gel when boiled in liquid and cooled to room temp. The Buffer Ions in solution Conduct the current from the electrodes through the gel Maintain pH so that biomolecules keep their original charge typical gels Movement of biomoleculesDNA, RNA, Proteins Strong electric field → large force moves molecules faster For nucleic acids (DNA and RNA), average voltages range from ~80-200 Volts DC Size (molecular weight) net charge Conformation (shape) Electrophoresis LAB Characterize physical features of dye molecules (relative size, charge, relative amount) Determine whether dyes are pure (only 1 color) or complex (more than one color) Identify the 2 dyes in an unknown mixture These are some of the individual colors in your dyes Blue #1 792.85 g/mol Terminology Pure dye has 1 color Complex dye has more than one color in it. Your unknown is made of 2 dyes (may be complex, pure or one of each) * Electrophoresis /ddnalc/resources/electrophoresis.html THE ELECTRIC FIELD + anode - cathode Parallel Electrodes Electric field (direction of conventional current) Electron flow + + - - The more concentrated the gel matrix, the slower the migration Goal: Maximize the separation of molecules of different molecular weights Minimize changes in electrolyte conductivity Don’t overheat (melt) the gel Physical features of biomolecules affect their ability to be separated from each other: DNA External Phosphates give DNA a net Negative charge. - - - - - - - - - - - - - Longer molecule means a lot more atoms and a few more negative charges. Larger molecules go slower Size and Net Charge As length increases, charge/mass ratio Decreases. Conformation (shape) DNA can be supercoiled -