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Class policies and expectationsOverall structure of class
Last class
Principle of DNA extractionRE Digests
Lab 1: Restriction enzyme cloning
Digest plasmid with RE1
Previous plasmid
Digest gene of interest (4Kb) with RE1“Ligate” gene (no RE1/2 sites) & plasmidPredicted 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
RE1: 3,2 Kb
Uncut: 5 Kb
RE2: 1,4 Kb
RE1+2: 0.5Kb, 1Kb, 4Kb
RE1: 4,3,2 Kb
Uncut: 9 Kb
RE2: 1,8 Kb
RE1+2: 0.5Kb, 1Kb, 7Kb
Conclusion?
Lab 1: Plasmid map assignment
TABLE (apprx.) fragment sizes
DUE Week 4
RE Map (with relative orientation of sites)RE Map does not require fragment sizesRE 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 lengthKeep 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 groupsHeme group absorbs at 418nm
Cyt P450 contains Heme
Can you tell which extract has Cyt P450?
Optical density – Uses
Measure enzyme activity
NADPH abs is maximal at 340nmAs reaction proceeds, OD340?
Fatty Acid + NADPH + O2
Hydroxylated fatty acid + NADP + H20
Cyt P450
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
Literature
Assumption
Ideas are correct
How /Method
Unimportant
Prior knowledge
Low requirement
Reading Passive
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
How does Dr. K read a paper?
Read Introduction – Identify main purposeLook at figures and figure legends
Read Title and Abstract (relatively quickly)
AFTER coming to MY conclusions, check author’s conclusionsIssues, 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 FALSEDo experiment, observe results
Results; Therefore “X” is TRUE/FALSE
Unexpected results/observations = Discovery!
A fundamental problem…
Want to study protein interactions
Want to study localization of proteins
In LIVE cells!
How to visualize proteins?
Seeing things
The modern repertoire
We want more!
“New” VS “Old”
Dynamics of proteins
Movement
Etc.