1. Lecture 3 Agarose Gel Electrophoresis 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. DNA is negatively charged +- Power DNA  When placed in an electrical field, DNA will migrate toward the positive pole (anode) HH O2O2 An agarose gel is used to slow the movement of DNA and separate by size Scanning Electron Micrograph of Agarose Gel (1×1 µm)  Polymerized agarose is porous, allowing for the movement of DNA

3. +- Power DNA 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. 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

5. Discovery of restriction enzymes meant 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 Restriction enzymes

6. DNA sequencing