1. Last class Separating and visualizing DNA Experimental design

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

3. Cloning a gene What is a “vector?” What is “cloning” a gene?

4. Cloning a gene PlasmidOpen plasmid Isolate gene Mix Ligate plasmid

5. Gene cloning Use __________________ to cut gene Use __________________ to open plasmid Use __________________ to ligate

6. Restriction endonucleases Type II REs “cut” inside ________________ Type II REs cut at ___________________________ sites _______________ = specific DNA sequence Almost always a palindromic sequence because: No length limits for RE site. Usually 4-8 nucleotides

7. Type II REs GCATGC CGTACG RE1 GC CGTA ATGC CG

8. The scientific process 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!

9. Reading a paper VS textbook TextbookLiterature Assumption Ideas are correct How /Method Unimportan t Prior knowledge Low requirement Reading Passive

10. Parts of a paper: Establish rationale Abstract/Summary Introduction

11. Parts of a paper: Present data Results Methods & Materials

12. Parts of a paper: Put in context, future, etc. Discussion References

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. Putting it all together 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 papers 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. Glucose–TOR signalling reprograms the transcriptome and activates meristems Building a knowledge base… “By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme.” eLife 2013;2:e00278 Nature 496, 181–186 Read, read, read, read, read.

18. Example of “figure reading” Main hypothesis: Yeast SUB1 (PC4) is required for NHEJ yku REQUIRED for NHEJ BamHI = RE Design experimental flow-chart (with controls!) to test hypothesis Eg: Generate dsDNA breaks -> ….

19. Example of “figure reading” Main hypothesis: Yeast SUB1 (PC4) is required for NHEJ yku REQUIRED for NHEJ BamHI = RE * = significantly different from WT (no other significant differences)

20. Figuring our figures… Main hypothesis: Yeast SUB1 (PC4) is required for NHEJ Objections? Next step/s?

21. The scientific process 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!

22. Lifecycle of C. elegans C. elegans Life Stages from George L. Sutphin and Matt Kaeberlein at Univ. of Washington, Seattle.

23. Lineage genes in C. elegans

24. lin-4 regulates lin-14

25. Lineage cascade in C. elegans

26. The lineage cascade in worms

27. Molecular mechanisms of lin-4 lin-14, lin-28, lin-29 were all proteins lin-4 = NOT protein! lin-4 in intron of another gene!

28. The scientific process 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!

29. So what is lin-4?! WTWT lin-4lin- 4(2) lin- 4(3)

30. The parts that we ignored

31. lin-4 is an RNA?!?! lin-4 is an RNA! lin-4 is a small (~22nt) RNA! How does small RNA regulate level of another protein?

32. Small RNAs = RNAi ++++

33. Paper 1 lin-4 & let-7  Only in C. elegans? Found let-7-like sequences in other organisms Small RNAs  Universal regulators? Small RNAs  How widespread? Roles?

34. Paper 1 assignment Download assignment Answer each question about indicated figure Do not go over space limit/Do not change font! Save/Convert to PDF Upload to Turnitin

35. Paper reading and Peer reviews Peer review is not a waste of time! Read lab reports critically ~ Reading paper! Gain: process, not just getting feedback Peer review = Universal career requirement!

36. Lab reports and Peer reviews Lab report one deadline varies Due before your lab section Peer review deadline: Calendar Cannot do reviews after deadline!!!