Last class Separating and visualizing DNA Experimental design.

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Presentation transcript:

Last class Separating and visualizing DNA Experimental design

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: Need DNA “scissors”!

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

Cloning a gene PlasmidOpen plasmid Isolate gene Mix Ligate plasmid

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

Restriction endonucleases Type II REs “cut” inside dsDNA Type II REs cut at RE site = Almost always a _______________ sequence because: No length limits for RE site. Usually ___ nucleotides

Type II REs GCATGC CGTACG RE1 GC CGTA ATGC CG

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!

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

Parts of a paper: Establish rationale Abstract/Summary Introduction

Parts of a paper: Present data Results Methods & Materials

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

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

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

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.

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!

Example of “figure reading” Main hypothesis: Yeast SUB1 (PC4) is required for NHEJ yku REQUIRED for NHEJ BamHI = 5’ overhang

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

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

Lineage genes in C. elegans

lin-4 regulates lin-14

Lineage cascade in C. elegans

The lineage cascade in worms

Molecular mechanisms of lin-4 lin-14, lin-28, lin-29 were all proteins lin-4 = lin-4

The parts that we ignored

lin-4 is an RNA?!?! lin-4 is lin-4 is a How does

Small RNAs = RNAi ++++

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?

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 (watch out for deadline!)

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!

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