Biol2 Lecture 2 Dna Isolation And Agarose Gel


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Lecture 2 - Biol 2

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  • Biol2 Lecture 2 Dna Isolation And Agarose Gel

    1. 1. DNA Isolation & Agarose Electrophoresis Lecture 2 Source:
    2. 2. <ul><li>Nucleic Acid purification can be performed starting from various biological materials: </li></ul><ul><ul><li>Blood </li></ul></ul><ul><ul><li>Tissues </li></ul></ul><ul><ul><li>Bacteria </li></ul></ul><ul><ul><li>Fungi </li></ul></ul><ul><ul><li>Animal cells </li></ul></ul><ul><ul><li>Plant cells </li></ul></ul><ul><ul><li>Dung/Stool </li></ul></ul><ul><ul><li>Agarose gels </li></ul></ul>
    3. 3. <ul><li>Type of starting material </li></ul><ul><li>Expected yield </li></ul><ul><li>Sample age (I.e. quality) </li></ul><ul><li>Time required for procedure </li></ul><ul><li>Cost </li></ul><ul><li>Quality control issues </li></ul>Technique used depends on …
    4. 4. General Extraction Protocols Include: <ul><li>Tissue Digestion / Cell Lysis </li></ul><ul><li>Chelating and Proteinase Agents </li></ul><ul><li>Nucleic Acid and Protein Separation </li></ul><ul><li>Clean-up </li></ul>
    5. 5. Tissue Digestion / Cell Lysis <ul><li>Disrupt cells or tissue </li></ul><ul><li>Avoid shearing forces that fragment DNA </li></ul><ul><li>Method used depends on cell type </li></ul><ul><li>Examples of methods: </li></ul><ul><ul><li>enzyme-based lysozymes </li></ul></ul><ul><ul><li>sonication </li></ul></ul><ul><ul><li>high-salt hypotonic bursting </li></ul></ul><ul><ul><li>SDS detergent membrane disruption </li></ul></ul>
    6. 6. QIAgen DNeasy DNA Kit <ul><li>Treat with Proteinase K </li></ul><ul><ul><ul><li>- digest proteins and inhibit nucleases </li></ul></ul></ul><ul><li>Lysis: QIAgen Buffer AL (guanidium-HCl) </li></ul><ul><ul><ul><li>- hypertonic high salt solution </li></ul></ul></ul><ul><li>Precipitation of DNA with EtOH </li></ul><ul><li>Spin column - DNA binds to silica membrane </li></ul><ul><li>Wash </li></ul><ul><li>Elution 1 and 2, with warm Buffer AE (Tris-EDTA) </li></ul>
    7. 7. Agarose Gel Electrophoresis <ul><li>Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. </li></ul>
    8. 8. <ul><li>DNA will be attracted to the positively charged pole ( RED ) and repelled from negatively charged pole (BLACK) </li></ul>“ Run to the Red” DNA is negatively charged because of the sugar-phosphate backbone.
    9. 9. Agarose <ul><li>Polymerizes to form a solid ‘gel’ which is porous allowing for the movement of DNA. </li></ul><ul><li>Small DNA fragments pass more easily through pores than large ones. </li></ul>D-galactose 3,6-anhydro L-galactose Scanning Electron Micrograph of Agarose Gel
    10. 10. An agarose gel is prepared by combining agarose powder and a buffer solution. Agarose  TBE Buffer  Flask for boiling  Special thanks to Michael Clark (University of Rochester) for these images.
    11. 11. Casting tray  Gel combs  Power supply  Gel tank   Cover Electrical leads  Electrophoresis Equipment
    12. 12. Seal the edges of the casting tray with masking tape and put in one comb Preparing the Casting Tray
    13. 13. Agarose 1x TBE Buffer Combine the agarose powder and 1x TBE buffer. Use a flask that is several times larger than the volume of buffer.
    14. 14. Melting the Agarose Insoluble at room temp Soluble once boiled Once the gel has reaches about 55 o C, Add 4-5 µ l of Ethidium Bromide dye to pre-stain.
    15. 15. Ethidium Bromide Warning! Mutagen! Intercalates into nucleic acid and fluoresces under UV light
    16. 16. Carefully pour the melted agarose solution into the casting tray. Avoid air bubbles. Pouring the gel
    17. 17. The gel comb should be submerged in the melted agarose solution.
    18. 18. When cooled (> 20 mins), the agarose polymerizes, forming a flexible gel. Carefully remove the combs and tape.
    19. 19. Place the gel in the electrophoresis tank (aka gel box). “ Run to the Red” Cathode (black -) Anode (red +)
    20. 20. TBE buffer  Add enough 1x TBE buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.
    21. 21. 6X Loading Buffer:  Bromophenol Blue (colour)  Glycerol (weight) Sample Preparation Mix the samples of DNA with the 6X sample loading buffer. This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.
    22. 22. Loading the Gel Carefully place the pipette tip inside a well and gently/slowly expel the sample. The sample should sink into the well.
    23. 23. Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. When the power is turned on, bubbles should form on the electrodes wires in the electrophoresis chamber. Running the Gel
    24. 24.  wells  Bromophenol Blue Cathode (-) Anode (+) After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA. DNA (-) 
    25. 25. Visualizing the DNA under UV light 1 2 3 4 2.0 0.6
    26. 26. Eric’s Quote of the Week <ul><li>‘ Never throw away your samples until the end of the experiment.’ </li></ul><ul><li>Dumpster diving SUCKS! </li></ul>Source: