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Last class Class policies, etc. VNTRs PCR Visualizing DNA DNA is not colored; Can’t see in solution. Separate pieces based on ____________ Run mixture through an __________ gel Agarose solution is like JellO. Liquid when hot, solid(ish) when cold - Agarose is NOT edible - Agarose tastes really, really bad Separating DNA Agarose gel is porous Small pores in gel allow DNA to pass through _______ DNA passes easily, _____ DNA less easily Agarose gel - + Agarose gel http://www.youtube.com/watch?v=2UQIoYhOowM Loading dye Why do you need a “buffer?” Why do you add glycerol? Why do you need the “dye”? Seeing the DNA Gel/buffer contains ETHIDIUM BROMIDE (EtBr) EtBr + UV light = ________ fluorescence EtBr in DNA + UV light = __________ fluorescence Regions in gel with DNA will show a BRIGHT “band” http://wikidoc.org/index.php/Agarose_gel_electrophoresis Estimating size of DNA 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 Changing gel parameters Wh y? Increase voltage Increase percentage of gel Increase time of run Watch out! LAB 2 Gel will be poured for you Load and run gel Check results Lab 3 onwards = pour your own gel 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 Do experiment, observe results FALSE Results; Therefore “X” is TRUE/FALSE Unexpected results/observations = Restriction endonucleases RE is an enzyme! - Optimal temperature - Optimal salt concentration/composition - Optimal pH - Time of activity RE cutting is random! Designing experiments Tube 10X Buffer RE (µL) (µL) Water 1N HCl (µL) (µL) 1 1.0 1.0 - 8.0 0.0 2 1.0 1.0 1.0 7.0 0.0 3 1.0 1.0 - 7.0 1.0 4 1.0 1.0 1.0 6.0 1.0 5 1.0 1.0 - 6.0 2.0 6 1.0 1.0 1.0 5.0 2.0 Designing experiments Tube DNA (µl) 10X Buffer RE (µL) (µL) Water 1N HCl (µL) (µL) 1 1.0 1.0 - 8.0 0.0 2 1.0 1.0 1.0 7.0 0.0 3 1.0 1.0 - 7.0 1.0 4 1.0 1.0 1.0 6.0 1.0 5 1.0 1.0 - 6.0 2.0 6 1.0 1.0 1.0 5.0 2.0 What variable is being tested? Draw What are controls? Additional controls? Designing experiments Form a hypothesis Design an experiment Predictions? LAB 2 Design experiment to test ONE variable - Time - Buffer - Enzyme concentration Discuss with partner, other groups & TA Generate experimental design Include controls! Interpreting results: Making predictions Effect of variable on RE digestion of linear 10Kb DNA Complete digestion with RE = 2,3, 5Kb bands Draw gel for 0%, 10%, 50% and 75% digestion