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Lecture 4 Agarose Gel Electrophoresis Ch. 2; 3

Lecture 4 Agarose Gel Electrophoresis Ch. 2; 3. Gel electrophoresis is a technique for the analysis of nucleic acids and proteins and preparation and analysis of DNA

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Lecture 4 Agarose Gel Electrophoresis Ch. 2; 3

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  1. Lecture 4 Agarose Gel ElectrophoresisCh. 2; 3 Gel electrophoresis is a technique for the analysis of nucleic acids and proteins and preparation and analysis of DNA Gel electrophoresis separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field We will be using agarose gel electrophoresis to determine the presence and size of PCR products.

  2. • When placed in an electrical field, DNA will migrate toward the positive pole (anode) H  O2  DNA - + Power • Polymerized agarose is porous, allowing for the movement of DNA Scanning Electron Micrograph of Agarose Gel (1×1 µm)  • DNA is negatively charged • An agarose gel is used to slow the movement of DNA and separate by size

  3. DNA - + Power How fast will the DNA migrate? • strength of the electrical field, buffer, density of agarose gel… • Size of the DNA! • *Small DNA move faster than large DNA • …gel electrophoresis separates DNA according to size small large • Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight

  4. Agarose D-galactose 3,6-anhydro L-galactose • Sweetened agarose gels have been eaten in the Far East since the 17th century • Agarose was first used in biology when Robert Koch* used it as a culture medium for Tuberculosis bacteria in 1882 • Lina Hesse, technician and illustrator for a colleague of Koch was the first to suggest agar for use in culturing bacteria Agarose is a linear polymer extracted from seaweed

  5. Restriction enzymes • What does a virus do? • They are natural defence mechanisms from viruses • First discovered in E-coli • eco-RI • One every 4000bp 5’-AGCTAGAATTCTTACC-3’ 3’-TCGATCTTAAGAATGG-5’ 5’--------3’OH AATT---------------------- 3’---------------TTAA5’ Palandrone Restriction site

  6. Restriction enzymes • Discovery of restriction enzymes mean a whole new list of analysis could be done • Cloning • DNA sequencing • Sanger method: 1977 • Labelled ddNTPs • Terminate DNA copying at random points • Product is labelled fragments varying in length

  7. DNA sequencing

  8. DNA sequencing

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