class policies and expectations overall structure of class last class principle of dna extraction re...

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.