Part 1. Gel electrophoresis DNA is negatively charged (because of phosphate backbone) DNA will be...
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Transcript of Part 1. Gel electrophoresis DNA is negatively charged (because of phosphate backbone) DNA will be...
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
• Serve as a natural defense mechanism for bacteria against viral infection
• Bacteria protect their DNA from cutting by their own enzymes through methylation
Examples
Enzyme Recognition sequence • EcoRI GAATTC• HindIII AAGCTT• BamHI GGATCC• EcoRV GATATC
• Recognition sequences are usually 4-8 base pairs in length and are usually palindromic
A closer look…. BamHI
5’….ACTGTACGGATCCGCTA….3’3’….TGACATGCCTAGGCGAT….5’
BamHI
A closer look…. BamHI
5’….ACTGTACG GATCCGCTA….3’3’….TGACATGCCTAG GCGAT….5’
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
5’….ACTGTACAGATCCGCTA….3’ 3’….TGACATGTCTAGGCGAT….5’
DNA Ligase
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
Gel electrophoresis
Agarose gel
- electrode + electrodeDNA fragments
buffer~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
Gel electrophoresis
- electrode + electrode
current
buffer~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~
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
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!!