Experiment 12 Agarose Gel Electrophoresis
Separation of DNA Fragments by Gel Electrophoresis <ul><li>Two Types </li></ul><ul><ul><li>Polyacrylamide gels </li></ul><...
<ul><li>Polyacrylamide Gels </li></ul><ul><ul><li>Separate DNA fragments up to 1000 bp long </li></ul></ul><ul><ul><li>Hig...
<ul><li>Agarose Gels </li></ul><ul><ul><li>Separate mixtures of fragments up to 20 kb </li></ul></ul><ul><ul><li>Easier to...
<ul><li>Agarose Gel Electrophoresis </li></ul><ul><ul><li>Carbohydrate polymer purified from salt water algae </li></ul></...
Bands of gel are visualized with UV light source <ul><li>central dye Ethidium Bromide </li></ul><ul><li>Fluorescent under ...
 
Results and Discussion <ul><li>Fragments of linear DNA migrate through agarose gels with a mobility that is inversely prop...
Results and Discussion <ul><li>Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the sam...
Results and Discussion <ul><li>Several additional factors have important effects on the mobility of DNA fragments in agaro...
Results and Discussion <ul><li>Agarose Concentration   </li></ul><ul><li>Higher concentrations of agarose facilitate separ...
Results and Discussion <ul><li>Larger fragments are much better resolved in the 0.7% gel </li></ul><ul><li>hile the small ...
Results and Discussion <ul><li>Voltage   </li></ul><ul><li>As the voltage applied to a gel is increased, larger fragments ...
Results and Discussion <ul><li>Electrophoresis Buffer   </li></ul><ul><li>Buffers not only establish a pH, but provide ion...
Results and Discussion <ul><li>Effects of Ethidium Bromide   </li></ul><ul><li>Binding of ethidium bromide to DNA alters i...
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Agarose Gel

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Agarose Gel

  1. 1. Experiment 12 Agarose Gel Electrophoresis
  2. 2. Separation of DNA Fragments by Gel Electrophoresis <ul><li>Two Types </li></ul><ul><ul><li>Polyacrylamide gels </li></ul></ul><ul><ul><li>Agarose gels </li></ul></ul>
  3. 3. <ul><li>Polyacrylamide Gels </li></ul><ul><ul><li>Separate DNA fragments up to 1000 bp long </li></ul></ul><ul><ul><li>Higher resolving power than agarose </li></ul></ul><ul><ul><li>Separate fragments that differ in length by single nucleotide </li></ul></ul>
  4. 4. <ul><li>Agarose Gels </li></ul><ul><ul><li>Separate mixtures of fragments up to 20 kb </li></ul></ul><ul><ul><li>Easier to prepare </li></ul></ul><ul><ul><li>Lower resolution </li></ul></ul>
  5. 5. <ul><li>Agarose Gel Electrophoresis </li></ul><ul><ul><li>Carbohydrate polymer purified from salt water algae </li></ul></ul><ul><ul><li>Copolymer of mannose and galactose </li></ul></ul><ul><ul><li>Melting and cooling form a gel with various pore sizes </li></ul></ul><ul><ul><li>Overall speed based on concentration of agarose </li></ul></ul><ul><ul><li>Pulsed-field gel electrophoresis </li></ul></ul><ul><ul><li>Migration of DNA </li></ul></ul><ul><ul><ul><li>Negatively charged phosphate backbone </li></ul></ul></ul><ul><ul><ul><li>size of DNA fragments </li></ul></ul></ul><ul><ul><li>Size of DNA fragments compared to standard </li></ul></ul>
  6. 6. Bands of gel are visualized with UV light source <ul><li>central dye Ethidium Bromide </li></ul><ul><li>Fluorescent under UV light </li></ul>
  7. 8. Results and Discussion <ul><li>Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. </li></ul>
  8. 9. Results and Discussion <ul><li>Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. </li></ul>
  9. 10. Results and Discussion <ul><li>Several additional factors have important effects on the mobility of DNA fragments in agarose gels </li></ul>
  10. 11. Results and Discussion <ul><li>Agarose Concentration </li></ul><ul><li>Higher concentrations of agarose facilitate separation of small DNAs, </li></ul><ul><li>Low agarose concentrations allow resolution of larger DNAs. </li></ul>
  11. 12. Results and Discussion <ul><li>Larger fragments are much better resolved in the 0.7% gel </li></ul><ul><li>hile the small fragments separated best in 1.5% agarose </li></ul>
  12. 13. Results and Discussion <ul><li>Voltage </li></ul><ul><li>As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments </li></ul>
  13. 14. Results and Discussion <ul><li>Electrophoresis Buffer </li></ul><ul><li>Buffers not only establish a pH, but provide ions to support conductivity. </li></ul><ul><li>DNA fragments will migrate at different rates due to differences in ionic strength. </li></ul>
  14. 15. Results and Discussion <ul><li>Effects of Ethidium Bromide </li></ul><ul><li>Binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility </li></ul>

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