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Isolation of Plasmid DNA

June 21, 2007Leeward Community College

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E. coliChromosome

Plasmids

Plasmids DNA molecules separate from chromosomal DNA Self-replicating

Electron micrograph of DNAfrom a lysed E. colicell

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F-plasmids: Facilitate bacterial conjugation

R-plasmids: Confer resistance to antibiotics or other toxins

Col-plasmids: Encode for colicines (potentially toxic to other bacteria)Degradative plasmids: Enable the breakdown of certain substancesVirulence plasmids: Causes the bacteria to act as a pathogen

Plasmid Functional Categories

Bacteria carrying a plasmid with the geneneomycin phosphotransferaseare capableof surviving in the presence of the antibiotickanamycin

http://en.wikipedia.org/wiki/Image:BacterConjugation.pnghttp://en.wikipedia.org/wiki/Image:BacterConjugation.pnghttp://en.wikipedia.org/wiki/Image:BacterConjugation.png

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Molecular Biology Applications for PlasmidsI. Cloning of DNA fragments

II. Protein Production

+ IPTGT7

promoter Gene of Interest

Protein Induction Plasmid

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Comparison of plasmid copy numbers in E. coli

High copy

DNA yield

Likelihood of mutation

Difficulty in cloning toxic genes

Low copy

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Gal4 DNA-bindingDomain

BaitProtein

Gal4 ActivationDomain

Bait Vector Prey Vector

LibraryProtein

Molecular Biology Applications for PlasmidsIII. Yeast Two-Hybrid Assay:

Tests for protein-protein interactions

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Molecular Biology Applications for PlasmidsIV. Agrobacterium-mediated Plant Transformation

A means of performing plant genetic engineering

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How to purify plasmid DNA usingsilica-based columns

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Harvest cells by centrifugation

Spin ~5,000 rcfSupernatant (clear)

Pelleted cellsE. coliculture(cloudy)

Discard supernatantResidual media may interfere with downstream steps

Resuspend cells in bufferThoroughly resuspend cells, making sure that noclumps remain. P1 buffer contains:

Tris-Cl (buffering agent) EDTA (metal chelator) RNase A (degrades RNA)

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Dissolves membranes

Binds to and denatures proteins

2. NaOH Denatures DNA

Because plasmids are supercoiled,both DNA strands remain entangledafter denaturation

Lyse cells with SDS/NaOH solution

Adding buffer P2 causes solution to become viscous

1. Sodium dodecyl sulfate

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Neutralize NaOH with potassium acetate solution

Mixing with buffer N3 causes a fluffy white precipitate toform.

1. Potassium acetate / acetic acid solution Neutralizes NaOH (renatures plasmid DNA)

Converts soluble SDS to insoluble PDS

sodium dodecyl sulfate (SDS) potassium dodecyl sulfate (PDS)(H2O sol. = 10%) (H2O sol. < 0.02%)

2. Guanidine hydrochloride (GuCl) Chaotropic salt; facilitates DNA binding to silica in

later steps

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Separate plasmid DNA from contaminants bycentrifugation

Supernatant contains:- Plasmid DNA

- Soluble cellular constituents

Pellet contains:- PDS- Lipids- Proteins

- Chromosomal DNA

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Add cleared lysate to column and centrifuge

The high ionic strength and presence of chaotropic saltcauses DNA to bind to the silica membrane, while othercontaminants pass through the column

Silica-gel membrane

Centrifuge

Flow through(discard)

Nucleic acids

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Wash the silica membrane to remove residualcontaminants

Buffer PB contains isopropanol and GuCl

Buffer PE contains ethanol and Tris-Cl

Centrifuge

PB + contaminants

Nucleic acids

Nucleic acids

PB buffer

Centrifuge

PE + contaminants(including residual GuCl)

Nucleic acids

Nucleic acids

PE buffer

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Elute purified DNA from the column

EB is 10 mM Tris-Cl (pH 8.5). TE or dH2O mayalso be used.

Centrifuge

EB + DNA

Nucleic acids

EB buffer

Buffer EB should be added directlyto the membrane for optimal DNArecovery and to avoid possibleEtOH contamination (from residualPE buffer)

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Manual alkaline lysis preparation of plasmid DNA

Organic extraction (optional)

Mix thoroughly withan equal volume oforganic solvent

e.g. phenol, chloroform,or phenol:chloroform

Centrifuge

Organic

Aqueous

Precipitate DNA with isopropanol (1:1 volume)Supernatant

Pellet

Centrifuge50% isopropanol+

precipitated DNA

Wash pellet with 70% EtOH (to remove salts) Dissolve pellet with TE (or other aqueous solution)

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Assessing your plasmid preparation

1. Quantify abundance (A260) and purity (A260/A280)

2. Verify by restriction digestion

3. Run undigested plasmid to see if it is mostlysupercoiled

supercoileddenatured