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Separating and visualizing DNA PowerPoint PPT Presentation

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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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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”!


What is a “vector?”

What is “cloning” a gene?

Cloning a DNA piece into a plasmid


Open plasmid

Isolate gene


Ligate plasmid


Use __________________ to cut gene

Use __________________to open plasmid

Use __________________to ligate

Restriction Enzymes (REs)

Enzyme that cuts DNA

Called a “Restriction” enzyme because:

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

Type II REs






What can I clarify?

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!

Reading primary literature

Primary means of scientific communication

- Build upon existing research

- Do not duplicate existing research

Sharing of ideas for scientific progress

Organization of a scientific paper





Methods & Materials


The parts of a paper: Establish rationale


- Brief version of paper

- Main question/s addressed

- Experimental approach

- Main result/s & their significance


- Background and context for the research

- Purpose/Hypothesis

The parts of a paper: Present data


- Data generated by experiments (Graphs, charts, tables, etc.)

- Explanations to understand results

Methods & Materials

- Science should be reproducible!

- Allows reader to duplicate results

The parts of a paper: Put in context, future


- Brief summary of results

- Significance

- Future directions


- Credit for past work

- Resource for additional information/reading

How to read primary literature

Not easy!

- Usually quite jargony

- Practice, practice, practice


- Each experiment will build on previous one

Active & Critical

- Research, not text book!

- Conclusions may not be correct!

Textbook VS Primary literature




Ideas are correct

Ideas are wrong!

How /Method



Prior knowledge

Low requirement






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.

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?


Main hypothesis: Yeast SUB1 (PC4) is required for NHEJ


BamHI = 5’ overhang

C. elegans life cycle

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

The lineage cascade in C. elegans

Molecular mechanisms

lin-14, lin-28, lin-29 were all proteins

lin-4 = NOT protein!

lin-4in intron of another gene!

But what does lin-4 encode?

Molecular mechanisms

lin-4 is an RNA!

lin-4is a small (~22nt) RNA!

How does small RNA regulate level of another protein?

Is lin-4 unique?

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

RNA interference mechanism = miRNA mechanism

Small RNAs generally regulate genes?

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?

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

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!

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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