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Separating and visualizing DNA

Last class. Separating and visualizing DNA. Experimental design. 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: - - -. First step:. Need DNA “scissors”!. Cloning.

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Separating and visualizing DNA

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  1. Last class Separating and visualizing DNA Experimental design

  2. 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: - - - First step: Need DNA “scissors”!

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

  4. Cloning a DNA piece into a plasmid Plasmid Open plasmid Isolate gene Mix Ligate plasmid

  5. How? Use __________________ to cut gene Use __________________to open plasmid Use __________________to ligate

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

  7. 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

  8. Type II REs RE1 GCATGCCGTACG RE1 GCCGTA ATGCCG

  9. What can I clarify?

  10. Reading primary literature Reviews, news, etc. ALWAYS biased! - News: Stem cells will allow us to live forever - Religious right: Billions of dollars wasted on useless, immoral research - Research: Stem cells MIGHT be therapeutic You might want to look at the actual research! Importance of forming your OWN opinions - Elections, democracy and political spin! - Informed decisions!

  11. Reading primary literature Primary means of scientific communication - Build upon existing research - Do not duplicate existing research Sharing of ideas for scientific progress

  12. Organization of a scientific paper Abstract/Summary Introduction Results Discussion Methods & Materials References

  13. The parts of a paper: Establish rationale Abstract/Summary - Brief version of paper - Main question/s addressed - Experimental approach - Main result/s & their significance Introduction - Background and context for the research - Purpose/Hypothesis

  14. The parts of a paper: Present data Results - Data generated by experiments (Graphs, charts, tables, etc.) - Explanations to understand results Methods & Materials - Science should be reproducible! - Allows reader to duplicate results

  15. The parts of a paper: Put in context, future Discussion - Brief summary of results - Significance - Future directions References - Credit for past work - Resource for additional information/reading

  16. How to read primary literature 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. Textbook VS Primary literature Textbook Literature Assumption Ideas are correct Ideas are wrong! How /Method Unimportant Critical! Prior knowledge Low requirement High requirement! Reading Passive Active!

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

  19. How to read a paper Main question/s or hypothesis/es of paper Individual figures - Purpose of the experiment - How does data relate to main question/s? Active & Critical - Does the data support conclusions? - Additional experiments? - Future directions - Controversy? Paradoxes?

  20. Example Main hypothesis: Yeast SUB1 (PC4) is required for NHEJ yku REQUIRED for NHEJ BamHI = 5’ overhang

  21. C. elegans life cycle C. elegans Life Stages from George L. Sutphin and Matt Kaeberlein at Univ. of Washington, Seattle.

  22. lineage genes in C. elegans

  23. lin-4 regulates lin-14

  24. The lineage cascade in C. elegans

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

  26. But what does lin-4 encode?

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

  28. Is lin-4 unique?

  29. 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?

  30. RNAi + + + +

  31. Paper 1 RNA interference mechanism = miRNA mechanism Small RNAs generally regulate genes?

  32. Paper 1 Found let-7-like sequences in other organisms Small RNAs  Universal regulators? Small RNAs  How widespread? Roles? How might you identify small RNAs?

  33. Your 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 Deadline: 18-Oct-2013 4:00pm

  34. Paper reading ~ Lab report reading! Peer review is nota waste of time! Read lab reports critically ~ Reading paper! Gain: process, not just getting feedback Peer review = Universal career requirement!

  35. Lab report 1 and Peer review Lab report one deadline varies Due before your lab section Peer review deadline is 01/28, 11:59pm Reviews released on 01/29, 12:00am Cannot do reviews after deadline!!!

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