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Last class Class policies and expectations Overall structure of class Principle of DNA extraction RE Digests Lab 1: Restriction enzyme cloning Previous plasmid Digest plasmid with RE1 Digest gene of interest (4Kb) with RE1 “Ligate” gene (no RE1/2 sites) & plasmid Predicted fragments with RE1 and RE2? Think about total size, orientation, change in RE sites Lab 1: Restriction enzyme cloning RE1 RE1 Lab 1: Restriction enzyme maps Uncut: 5 Kb Uncut: 9 Kb RE1: 3,2 Kb RE1: 4,3,2 Kb RE2: 1,4 Kb RE2: 1,8 Kb RE1+2: 0.5Kb, 1Kb, 4Kb RE1+2: 0.5Kb, 1Kb, 7Kb Conclusion? Lab 1: Plasmid map assignment DUE Week 4 TABLE (apprx.) fragment sizes RE Map (with relative orientation of sites) RE Map does not require fragment sizes RE Map should be presentable! Optical density – General theory Baseline: Extinction coefficient (E) Path length (l) Concentration (c) Optical density – General theory OD ∝ Concentration, OD ∝ Path length Keep Path length constant OD ∝ ________________ Manual: Details of Beer-Lambert’s law OD = _______ Optical density – Uses Protein concentration Dye (Coomassie Brilliant Blue G-250) Dye binds protein, Abs increases (at 595nm) More protein = ? OD = 2.5. [Protein] = ? Optical density – Uses Presence of specific groups Heme group absorbs at 418nm Cyt P450 contains Heme Can you tell which extract has Cyt P450? Optical density – Uses Measure enzyme activity Fatty Acid + NADPH + O2 Cyt P450 Hydroxylated fatty acid + NADP + H20 NADPH abs is maximal at 340nm As reaction proceeds, OD340? Using OD… I have a solution containing Nucleotides, Water, Salts, Plasmid DNA and DNA polymerase. I want to estimate [Plasmid DNA] by measuring OD260 (DNA absorbs at 260nm). What should I use as a Blank? “Analytical reading” (or “How to read a paper) Textbook Assumption Ideas are correct How /Method Unimportant Prior knowledge Low requirement Reading Passive Literature How does Dr. K read a paper? Read Title and Abstract (relatively quickly) Read Introduction Identify main purpose/hypothesis Look at figures and figure legends How does Dr. K “look” at figures? Figure out the experimental rationale, design Make predictions Look at figures – what are the results? Predictions VS Results -> Do I believe it? Missing information? Data? Controls? Each individual figure <-> Main purpose How does Dr. K read a paper? Read Title and Abstract (relatively quickly) Read Introduction – Identify main purpose Look at figures and figure legends AFTER coming to MY conclusions, check author’s conclusions Issues, controversy, applications, etc. Reading literature = Needs practice Not easy! - Usually quite jargony - Practice, practice, practice Sequential - Each experiment will build on previous one Active & Critical - Research, not text book! - Conclusions may not be correct! Science = 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! A fundamental problem… Want to study localization of proteins Want to study protein interactions In LIVE cells! How to visualize proteins? Seeing things The modern repertoire We want more! Dynamics of proteins “New” VS “Old” Movement Etc.