1. Class policies and expectations Overall structure of class Last class Principle of DNA extraction RE Digests

2. Lab 1: Restriction enzyme cloning Digest plasmid with RE1 Previous plasmid 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

3. Lab 1: Restriction enzyme cloning RE1

4. Lab 1: Restriction enzyme maps RE1: 3,2 Kb Uncut: 5 Kb RE2: 1,4 Kb RE1+2: 0.5Kb, 1Kb, 4Kb RE1: 4,3,2 Kb Uncut: 9 Kb RE2: 1,8 Kb RE1+2: 0.5Kb, 1Kb, 7Kb Conclusion?

5. Lab 1: Plasmid map assignment TABLE (apprx.) fragment sizes DUE Week 4 RE Map (with relative orientation of sites) RE Map does not require fragment sizes RE Map should be presentable!

6. Optical density – General theory Baseline: Extinction coefficient (E) Path length (l) Concentration (c)

7. Optical density – General theory Keep Path length constant Manual: Details of Beer-Lambert’s law OD = _______

8. Optical density – Uses Protein concentration Dye (Coomassie Brilliant Blue G-250) Dye binds protein, Abs increases (at 595nm) More protein = ? OD = 2.5. [Protein] = ?

9. Optical density – Uses Presence of specific groups Heme group absorbs at 418nm Cyt P 450 contains Heme Can you tell which extract has Cyt P 450 ?

10. Optical density – Uses Measure enzyme activity NADPH abs is maximal at 340nm As reaction proceeds, OD 340 ? Fatty Acid + NADPH + O 2 Hydroxylated fatty acid + NADP + H 2 0 Cyt P 450

11. Using OD… I have a solution containing Nucleotides, Water, Salts, Plasmid DNA and DNA polymerase. I want to estimate [Plasmid DNA] by measuring OD 260 ( DNA absorbs at 260nm ). What should I use as a Blank?

12. “Analytical reading” ( or “How to read a paper ) TextbookLiterature Assumption Ideas are correct How /Method Unimportant Prior knowledge Low requirement Reading Passive

13. How does Dr. K read a paper? Read Introduction Identify main purpose/hypothesis Read Title and Abstract (relatively quickly) Look at figures and figure legends

14. How does Dr. K “look” at figures? Make predictions Look at figures – what are the results? Figure out the experimental rationale, design Predictions VS Results -> Do I believe it? Missing information? Data? Controls? Each individual figure Main purpose

15. How does Dr. K read a paper? Read Introduction – Identify main purpose Look at figures and figure legends Read Title and Abstract (relatively quickly) AFTER coming to MY conclusions, check author’s conclusions Issues, controversy, applications, etc.

16. 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!

17. 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!

18. A fundamental problem… Want to study protein interactions Want to study localization of proteins In LIVE cells! How to visualize proteins?

19. Seeing things

20. The modern repertoire

21. We want more! “New” VS “Old” Dynamics of proteins Movement Etc.