Last class
Separating and visualizing DNA
Experimental design
Visualizing 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 a gene
What is a “vector?”
What is “cloning” a gene?
Cloning a gene
Plasmid
Open 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
RE1
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
Textbook
Assumption
Ideas are correct
How /Method
Unimportant
Prior
knowledge
Low requirement
Reading
Passive
Literature
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 Title and Abstract (relatively quickly)
Read Introduction
Identify main purpose/hypothesis
Look at figures and figure legends
How does Dr. K look at figures
Figure out the experimental rationale,
design
Make predictions
Look at figures – what are the results?
Predictions VS Results -> Do I believe it?
Missing information? Data? Controls?
Each individual figure <-> Main purpose
Putting it all together
Read Title and Abstract (relatively quickly)
Read Introduction – Identify main purpose
Look at figures and figure legends
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,
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
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+
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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!!!