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Part 1. Gel electrophoresis

Part 1. Gel electrophoresis. DNA is negatively charged (because of phosphate backbone) DNA will be attracted to positively charged poles and repelled from negatively charged ones. Part 2. Restriction Endonucleases (aka restriction enzymes). Enzymes that “cut” DNA in a sequence-specific manner

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Part 1. Gel electrophoresis

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  1. Part 1. Gel electrophoresis • DNA is negatively charged (because of phosphate backbone) • DNA will be attracted to positively charged poles and repelled from negatively charged ones

  2. Part 2. Restriction Endonucleases (aka restriction enzymes) • Enzymes that “cut” DNA in a sequence-specific manner • Serve as a natural defense mechanism for bacteria against viral infection • Bacteria protect their DNA from cutting by their own enzymes through methylation

  3. Examples EnzymeRecognition sequence • EcoRI GAATTC • HindIII AAGCTT • BamHI GGATCC • EcoRV GATATC • Recognition sequences are usually 4-8 base pairs in length and are usually palindromic

  4. A closer look…. BamHI BamHI 5’….ACTGTACGGATCCGCTA….3’ 3’….TGACATGCCTAGGCGAT….5’

  5. A closer look…. BamHI 5’….ACTGTACG GATCCGCTA….3’ 3’….TGACATGCCTAG GCGAT….5’

  6. Ligations • When DNA molecules with sticky ends come together, only hydrogen bonds between complimentary nucleotides are reformed • These H-bonds are not stable enough to be permanent • DNA ligase=enzyme that joins the ends of DNA and re-establishes the phosphodiester bond in the DNA molecule

  7. DNA Ligase 5’….ACTGTACAGATCCGCTA….3’ 3’….TGACATGTCTAGGCGAT….5’

  8. Running a gel • Molten agarose is poured into a casting tray and a comb is placed • After the agarose solidifies, the comb is removed leaving wells where the DNA will be loaded • DNA samples are mixed with tracking dye which contains sucrose (to weigh down the DNA) and dyes so that you can visualize migration • A buffer containing ions (to conduct an electric current) is placed in the chamber around the gel

  9. Gel electrophoresis - electrode DNA fragments + electrode Agarose gel ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

  10. Gel electrophoresis - electrode + electrode current ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

  11. Movement of DNA fragments in agarose gels • There is a linear relationship between the migration rate of a given DNA fragment and the logarithm of its size (in basepairs). • Larger molecules move more slowly through the gel because of more friction

  12. An ethidium-stained gel photographed under UV light **Each band that you see is a collection of millions of DNA molecules, all of the same length!!

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