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Electrophoresis of DNA

Electrophoresis of DNA. What is it used for? How does it work? How do we do it? What are the results?. What is electrophoresis?. Separation of charged molecules DNA has net negative charge. -. +. What is Agarose?. A polysaccharide polymer Prepared from seaweed Highly purified

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Electrophoresis of DNA

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  1. Electrophoresis of DNA • What is it used for? • How does it work? • How do we do it? • What are the results?

  2. What is electrophoresis? • Separation of charged molecules • DNA has net negative charge - +

  3. What is Agarose? • A polysaccharide polymer • Prepared from seaweed • Highly purified • Very water soluble • Creates a gel • Gel “pore” size depends on concentration of agarose

  4. What is Agarose Gel Electrophoresis? - - - - - - - - - • Larger DNA fragments migrate less • Smaller DNA fragments migrate more + + + + + + +

  5. What is Agarose Gel Electrophoresis? - • Larger DNA fragments migrate less • Smaller DNA fragments migrate more +

  6. What is Agarose Gel Electrophoresis? Agarose Gel Animation

  7. Pouring a gel • Calculate agarose amount (0.5-3%) • Measure agarose and add to buffer • Melt in microwave • Pour • Let cool • Remove comb

  8. Preparing the gel for loading • Place gel and tray into “submarine” chamber • Fill with buffer

  9. Preparing the DNA for loading • DNA must be added to a “loading dye” • Necessary because: • DNA must sink • Need to track samples • Loading dye contains: • Dye • High density components (Ficoll, glycerol). DNA (40 ul) DYE (10 ul) DNA Ready-to-Load (load 20 ul)

  10. Loading a gel Load “underwater” Let DNA flow from tip

  11. Running a gel – Part 1 • Connect to power supply • Verify polarity! • Voltage, time, current • Run!

  12. Running a gel – Part 2 • Connect to power supply • Verify polarity! • Voltage, time, current • Run! • Stain with ethidium bromide or dye Gel running

  13. Analyzing the results • Visualize bands (UV or visible light) • Compare to standard • Determine what DNA fragments are in your samples

  14. Troubleshooting • Bands run wrong direction • Fuzzy bands • Distorted bands • Faint or no bands • Bands crooked

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