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Demystifying Electrophoresis

Demystifying Electrophoresis. Electrophoresis. Used in biotechnology as a separation method Molecules move by attraction to an electric charge The porous gel allows small molecules to move faster than large molecules

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Demystifying Electrophoresis

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  1. Demystifying Electrophoresis

  2. Electrophoresis • Used in biotechnology as a separation method • Molecules move by attraction to an electric charge • The porous gel allows small molecules to move faster than large molecules • Applications include sorting mixtures of proteins or DNA molecules for identification purposes

  3. Equipment

  4. Power Supply

  5. Power Supplies Units • Units may have variable voltage or two fixed levels: 75 volts and 125 volts • Some have timers that keep track of running time • Variable voltage units can be used to measure current in milliamps (we use this feature for proving Ohm’s law)

  6. Gel Box

  7. Reagents

  8. Common Elements of Box • Positive end (red) & negative end (black) • Platinum wires at each end on bottom of gel box in “wells” (may be visible, may be covered) • Leads (black & red) that attach to power source • Platform for tray that holds gel

  9. Casting Tray • Can be used for holding gel in box • Slots to accommodate a comb with teeth that make the wells in the gel • Comb can be placed at the end (negative for DNA) or in the middle (for materials of unknown electric charge) • Dams are placed on ends to block molten agarose when pouring gels (may be rubber, plastic, or simply tape)

  10. Casting Tray

  11. Gel Concentration • The higher the concentration of agarose, the more it retards movement • High concentrations for small DNA fragments (1.0-1.2%) • Low concentrations for large fragments (0.8%) • Gels are made with different buffers for proteins and DNA • TBE or TAE – DNA • SDS – Proteins • NOT WATER (It wouldn’t conduct electricity!)

  12. Equipment Clean-up • Buffers are salty, so after use rinse with tap water & De-ionized or Distilled water • Only air dry the box, DO NOT Towel dry – as you might break the Platinum wire in each well (after rinse with De-I, there should be no water spots)

  13. Steps in Electrophoresis • Place gel in chamber (with or without gel tray – depending on unit) • Add appropriate buffer to chamber – approximately 150 ml of 1X buffer used to make the gels • Wells in gels should be visible in gel and completely submerged in buffer solution • Load your DNA samples into wells • Set lid on chamber and slide it shut • Plug leads into red and black outlets in power supply (red-red/black-black) • Turn power supply on and adjust voltage to desired speed • Stop Electrophoresis

  14. Voltage and Running time voltagetime 10V 20-24 hrs 20V 12-16 hrs 60V 2 hrs 120V 50 min

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