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Class policies and expectations
Overall structure of class
Principle of DNA extraction
RE Digests
Lab 1: Restriction enzyme cloning
Previous plasmid
Digest plasmid with RE1
Digest gene of interest (4Kb) with RE1
“Ligate” gene (no RE1/2 sites) & plasmid
Predicted fragments with RE1 and RE2?
Think about total size, orientation, change in RE sites
Lab 1: Restriction enzyme cloning
RE1
RE1
Lab 1: Restriction enzyme maps
Uncut: 5 Kb
Uncut: 9 Kb
RE1: 3,2 Kb
RE1: 4,3,2 Kb
RE2: 1,4 Kb
RE2: 1,8 Kb
RE1+2: 0.5Kb, 1Kb, 4Kb
RE1+2: 0.5Kb, 1Kb, 7Kb
Conclusion?
Lab 1: Plasmid map assignment
DUE Week 4
TABLE (apprx.) fragment sizes
RE Map (with relative orientation of sites)
RE Map does not require fragment sizes
RE Map should be presentable!
Optical density – General theory
Baseline:
Extinction coefficient (E)
Path length (l)
Concentration (c)
Optical density – General theory
OD ∝ Concentration, OD ∝ Path length
Keep Path length constant
OD ∝ ________________
Manual: Details of Beer-Lambert’s law
OD = _______
Optical density – Uses
Protein concentration
Dye (Coomassie Brilliant Blue G-250)
Dye binds protein, Abs increases (at 595nm)
More protein = ?
OD = 2.5. [Protein] = ?
Optical density – Uses
Presence of specific groups
Heme group absorbs at 418nm
Cyt P450 contains Heme
Can you tell which extract has Cyt P450?
Optical density – Uses
Measure enzyme activity
Fatty Acid + NADPH + O2
Cyt P450
Hydroxylated fatty acid + NADP + H20
NADPH abs is maximal at 340nm
As reaction proceeds, OD340?
Using OD…
I have a solution containing Nucleotides, Water, Salts,
Plasmid DNA and DNA polymerase.
I want to estimate [Plasmid DNA] by measuring OD260
(DNA absorbs at 260nm).
What should I use as a Blank?
“Analytical reading” (or “How to read a paper)
Textbook
Assumption
Ideas are correct
How /Method
Unimportant
Prior
knowledge
Low
requirement
Reading
Passive
Literature
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
How does Dr. K read a paper?
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 literature = Needs practice
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!
Science = Life!
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!
A fundamental problem…
Want to study localization of proteins
Want to study protein interactions
In LIVE cells!
How to visualize proteins?
Seeing things
The modern repertoire
We want more!
Dynamics of proteins
“New” VS “Old”
Movement
Etc.