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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.png8/4/2019 CY2007 Plasmid Isolation
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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
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