this week in lab pdb/rasmol homeworkww2.chemistry.gatech.edu › class › peek › 4581 › notes...

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Week #2 CHEM 4581 - Spring 2008 1

This Week in Lab

Lab Tour

Experiments– Pipettor Use and Calibration– DNA Concentration Determination– Bradford Protein Concentration Assay

Short Report Composition– Lab Report due in class– Use computer cluster or laptops

**REMINDER**Bring lab notebook

Bring safety glassesWear appropriate attire


PDB/RasMol Homework

Torsion Angles and their measurement– Psi and Phi angle definitions– Ramachandran plot

Source: Wikipedia


PDB/RasMol Homework

Hydrogen Bonds– Special type of dipole-dipole interaction– Heteroatoms Source: Wikipedia


DNA Plasmid Purificationand

Restriction Digestion

Characteristics of DNA plasmids DNA purification strategies Restriction enzymes Restriction mapping

Lecture Outline


What is a DNA Plasmid? Double-stranded DNA Extra-chromosomal Size: 1 -200 kbases Covalently closed, circular,

superhelical Bacterial Dependent on host cell’s

proteins for replication andtranscription machinery


Features of a DNA Plasmid

• Selectable Markers Ex: Amp Resistance

• Origin of Replication

• Bacteriophage Promoter T7 RNA Polymerase

Notice nomenclature:“pYP” or “pBR322”

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DNA Plasmid “pYP”


Scale of PurificationSmall: “Mini-Prep”

– 1-10 mL– 3-5 µg yield

Medium: “Midi-Prep” (<20 kb)– 10-100 mL– 10-200 µg yield

Large: “Maxi-Prep”– ~250 mL– 1 mg yield


Overexpression of pYP

Sterilize Luria-Bertani (LB) agar and broth– Tryptone– NaCl– Yeast extract

Cool then add ampicillin

Pour and streak agar plates

Grow @ 37 °C for 12-16 h.

Day 1



Isolate a colony

Inoculate starter culturecontaining ampicillin

Grow @ 37 °C for 8 h

Inoculate large culture overnight

Reference: Wikipedia

Day 2



Discard supernatant

Freeze pellet of E. coli cells

Each student receives pellet of E. coli cellsequivalent to 100 mL of culture

Day 3 - Harvest Cells


DNA Purification

NaOH - denaturant SDS - detergentAlkaline Lysis

Other Approaches?

Lysozyme + EDTA + SDS

Heat (i.e. boiling)

Sonication

Open Cell Wall

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DNA Plasmid Purification

Silica Based Adsorption

SeparationReservoir

EmptyColumn

1. Mix the cell lysate with the resin

2. Pour onto empty column


DNA Plasmid Purification

Silica Based Adsorption

Separation

3. Elute wash effluent with vacuum manifold

Ion Exchange Chromatography

Alternative approaches


Overview of Experiment

DNA Purification

Determine [DNA]– 1 OD = 50 µg/mL ds DNA– 1 OD = 38 µg/mL RNA

Restriction Digestion

Agarose Gel Electrophoresis


EcoR I bound to

DNA

Courtesy of Protein Data Bank (1QPS) - Rosenberg Lab

EcoR I Structure


Restriction Enzymes

Proteins that cleave ds DNA (4-8 bp segments)

Found in bacteria

Name indicates source microorganism– E coli Escherichia coli (Name of Bacterium)

– Ear I Enterobacter aerogenes (Name of Restriction Enzyme)


Role in Nature: Defense

Source: Sigma Aldrich

Methylate C3

MethylateN4

Could also Methylate N6 of Adenine

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Types of Restriction Enzymes

Type Activity Cleavage Site I Endonuclease & ≥ 1000 bp from

Methylase recognition sequence

II Endonuclease Within or nearrecognition sequence

III Endonuclease & ~24-26 bp fromMethylase recognition sequence


Type II Restriction Enzymes

Palindromic Recognition Sequences– “Madam, I’m Adam”– “Race Car”

EcoR I 5’-G-A-A-T-T-C-3’3’-C-T-T-A-A-G-5’

EcoR V 5’-G-A-T-A-T-C-3’3’-C-T-A-T-A-G-5’


Type II Restriction Enzymes

Sticky vs. Blunt Ended Cuts

EcoR I 5’-G-A-A-T-T-C-3’3’-C-T-T-A-A-G-5’

EcoR V 5’-G-A-T-A-T-C-3’3’-C-T-A-T-A-G-5’

Sticky Ends Formed Blunt Ends Formed

Isochisomers: restriction enzymes that recognize the same sequence


New England Biolabs

http://rebase.neb.com/rebase/rebase.html

http://tools.neb.com/NEBcutter2/index.php

this week in lab pdb/rasmol homeworkww2.chemistry.gatech.edu › class › peek › 4581 › notes...

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