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Visualizing DNA on Agarose Gels: Techniques and Protocols

Learn how to visualize DNA fragments on agarose gels, separate and analyze DNA based on size, and understand essential techniques including PCR and VNTRs. Discover the significance of buffers, dyes, size markers, and the role of agarose gel electrophoresis in genetic research.

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Visualizing DNA on Agarose Gels: Techniques and Protocols

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  1. Last class Class policies, etc. VNTRs PCR

  2. Visualizing DNA DNA is not colored; Can’t see in solution. Separate pieces based on size Run mixture through an “agarose” gel Agarose solution is like JellO. Liquid when hot, solid(ish) when cold - Agarose is NOT edible - Agarose tastes really, really bad

  3. Visualizing DNA Agarose gel is porous Small pores in gel allow DNA to pass through Smaller DNA passes easily, larger DNA less easily

  4. Agarose gels - +

  5. Agarose gels http://www.youtube.com/watch?v=2UQIoYhOowM

  6. Important points of agarose gels Why do you need a “buffer?” - - - Why do you add glycerol? - Why do you need the “dye”? -

  7. How to see DNA bands Gel/buffer contains ETHIDIUM BROMIDE (EtBr) EtBr in DNA + UV light = HIGH fluorescence EtBr + UV light = low fluorescence Regions in gel with DNA will show a BRIGHT “band” http://wikidoc.org/index.php/Agarose_gel_electrophoresis

  8. Size markers/DNA ladders See DNA. But size? Run DNA fragments of known length Distance run = size of DNA (compare to ladder) 2000 bp 1000 bp 600 bp 500 bp 400 bp 300 bp 200 bp http://wikidoc.org/index.php/Agarose_gel_electrophoresis

  9. Changing gel running conditions Increase voltage – run quicker… but… Increase percentage of gel - … Increase time of run – run longer …

  10. Lab 2 Gel will be poured for you Load and run gel Check results Lab 3 onwards = pour your own gel

  11. Working with DNA Genes/regions of interest in larger pieces of DNA Isolate gene/region of interest to study it/products Examples of things you can do with DNA: - Clone - Amplify - Mutate First step: Isolate or cut out gene/DNA of interest Need DNA “scissors”!

  12. Restriction Enzymes (REs) Enzyme that cuts DNA Called a “Restriction” enzyme because:

  13. Type II REs Type II REs “cut” inside dsDNA Type II REs cut at specific recognition sites RE site = specific DNA sequence Almost always a palindromic sequence because: No length limits for RE site. Usually 4-8 nucleotides

  14. How do we study life? I think “X” BECAUSE “Y” Design an experiment to PROVE “X” Experiment: Predict outcomes if “X” is TRUE Predict outcomes if “X” is FALSE Do experiment, observe results Results; Therefore “X” is TRUE/FALSE Unexpected results/observations = Discovery!

  15. RE activity can be regulated RE is an enzyme! - Optimal temperature - Optimal salt concentration/composition - Optimal pH - Time of activity RE cutting is random!

  16. Example of experimental design

  17. Experimental design Form a hypothesis Design an experiment Predictions?

  18. Lab 2 Design experiment to test ONE variable - Time - Buffer - Enzyme concentration Discuss with partner, other groups & TA Generate experimental design Include controls!

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