An Introduction to PCR：PCR导论.pptVIP

Electrophoresis of DNA What is it used for? How does it work? How do we do it? What are the results? What is electrophoresis? Separation of charged molecules DNA has net negative charge What is Agarose? A polysaccharide polymer Prepared from seaweed Highly purified Very water soluble Creates a gel Gel “pore” size depends on concentration of agarose What is Agarose Gel Electrophoresis? Larger DNA fragments migrate less Smaller DNA fragments migrate more What is Agarose Gel Electrophoresis? Larger DNA fragments migrate less Smaller DNA fragments migrate more What is Agarose Gel Electrophoresis? Pouring a gel Calculate agarose amount (0.5-3%) Measure agarose and add to buffer Melt in microwave Pour Let cool Remove comb Preparing the gel for loading Place gel and tray into “submarine” chamber Fill with buffer Preparing the DNA for loading DNA must be added to a “loading dye” Necessary because: DNA must sink Need to track samples Loading dye contains: Dye High density components (Ficoll, glycerol). Loading a gel Running a gel – Part 1 Connect to power supply Verify polarity! Voltage, time, current Run! Running a gel – Part 2 Connect to power supply Verify polarity! Voltage, time, current Run! Stain with ethidium bromide or dye Analyzing the results Visualize bands (UV or visible light) Compare to standard Determine what DNA fragments are in your samples Troubleshooting Bands run wrong direction Fuzzy bands Distorted bands Faint or no bands Bands crooked * * - + - - - - - - - - - + + + + + + + - + Agarose Gel Animation DNA (40 ul) DYE (10 ul) DNA Ready-to-Load (load 20 ul) Load “underwater” Let DNA flow from tip Gel running