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Electrophoresis

A tool for s eparating specific biomolecules from a mixture (A key step in most biotechnology applications). Electrophoresis. Electrophoresis: movement of charged particles through a substance when an electric field is applied. http://www.dnalc.org/ddnalc/resources/electrophoresis.html.

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Electrophoresis

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  1. A tool for separating specific biomolecules from a mixture(A key step in most biotechnology applications) Electrophoresis

  2. Electrophoresis: movement of charged particles through a substance when an electric field is applied. http://www.dnalc.org/ddnalc/resources/electrophoresis.html

  3. The Gel Substance • Agarose (from seaweed) • Polyacrylamide • Powders that gel when boiled in liquid and cooled to room temp.

  4. The Buffer • Ions in solution • Conduct the current from the electrodes through the gel • Maintain pH so that biomolecules keep their original charge

  5. THE ELECTRIC FIELD Parallel Electrodes - cathode Electric field (direction of conventional current) Electron flow + anode

  6. typical gels - - + +

  7. 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 • The more concentrated the gel matrix, the slower the migration

  8. Goal: • Maximize the separation of molecules of different molecular weights • Minimize changes in electrolyte conductivity • Don’t overheat (melt) the gel

  9. Physical features of biomolecules affect their ability to be separated from each other: • Size (molecular weight) • net charge • Conformation (shape)

  10. Size and Net Charge External Phosphates give DNA a net Negative charge. DNA - Longer molecule means a lot more atoms and a few more negative charges. - - - - - - As length increases, charge/mass ratio Decreases. - - - - - - Larger molecules go slower

  11. Conformation (shape) DNA can be supercoiled - goes faster than when it is linear Proteins can take on many different 3-D shapes – These are denatured in the gel so that they are all essentially linear.

  12. FElectric Ffriction Q+ ∑F = FE + Ff = ma = 0 Biomolecules migrate at Terminal velocity Who can resist a little Physics?: Force diagram + anode - cathode

  13. Electrolysis Water is H20 → H+ + OH- + anode - cathode e- e- 2H+ + 2e-→H2(gas) 2H2O→ O2(gas) + 4H+ + 4e- A little Chemistry: • The generated amount of hydrogen is twice the amount of oxygen. • Both are proportional to the total electrical charge that was sent through the water.

  14. 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

  15. These are some of the individual colors in your dyes Crystal Violet 408.0 g/mol Red #40 496.42 g/ mol Blue #1 792.85 g/mol Fluorescein 376.27 g/mol Yellow #5 452.38 g/mol Safranin 350.85 g.mol

  16. 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)

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