Isolation of Plasmid DNA June 21 2007 Leeward
- Slides: 17
Isolation of Plasmid DNA June 21, 2007 Leeward Community College
Plasmids E. coli Chromosome Electron micrograph of DNA from a lysed E. coli cell Plasmids • DNA molecules separate from chromosomal DNA • Self-replicating
Plasmid Functional Categories • F-plasmids: Facilitate bacterial conjugation • R-plasmids: Confer resistance to antibiotics or other toxins Bacteria carrying a plasmid with the gene neomycin phosphotransferase are capable of surviving in the presence of the antibiotic kanamycin • Col-plasmids: Encode for colicines (potentially toxic to other bacteria) • Degradative plasmids: Enable the breakdown of certain substances • Virulence plasmids: Causes the bacteria to act as a pathogen
Molecular Biology Applications for Plasmids I. Cloning of DNA fragments II. Protein Production + IPTG
Comparison of plasmid copy numbers in E. coli High copy DNA yield Likelihood of mutation Difficulty in cloning toxic genes Low copy
Molecular Biology Applications for Plasmids III. Yeast Two-Hybrid Assay: Tests for protein-protein interactions Gal 4 DNA-binding Domain Bait Protein Bait Vector Gal 4 Activation Domain Library Protein Prey Vector
Molecular Biology Applications for Plasmids IV. Agrobacterium-mediated Plant Transformation A means of performing plant genetic engineering
How to purify plasmid DNA using silica-based columns
Harvest cells by centrifugation Spin ~5, 000 rcf Supernatant (clear) E. coli culture (cloudy) Pelleted cells Discard supernatant Residual media may interfere with downstream steps Resuspend cells in buffer Thoroughly resuspend cells, making sure that no clumps remain. P 1 buffer contains: • Tris-Cl (buffering agent) • EDTA (metal chelator) • RNase A (degrades RNA)
Lyse cells with SDS/Na. OH solution Adding buffer P 2 causes solution to become viscous 1. Sodium dodecyl sulfate • Dissolves membranes • Binds to and denatures proteins 2. Na. OH • Denatures DNA Because plasmids are supercoiled, both DNA strands remain entangled after denaturation
Neutralize Na. OH with potassium acetate solution Mixing with buffer N 3 causes a fluffy white precipitate to form. 1. Potassium acetate / acetic acid solution • Neutralizes Na. OH (renatures plasmid DNA) • Converts soluble SDS to insoluble PDS 2. Guanidine hydrochloride (Gu. Cl) • Chaotropic salt; facilitates DNA binding to silica in later steps
Separate plasmid DNA from contaminants by centrifugation Supernatant contains: - Plasmid DNA - Soluble cellular constituents Pellet contains: - PDS - Lipids - Proteins - Chromosomal DNA
Add cleared lysate to column and centrifuge Centrifuge Nucleic acids Silica-gel membrane Flow through (discard) The high ionic strength and presence of chaotropic salt causes DNA to bind to the silica membrane, while other contaminants pass through the column
Wash the silica membrane to remove residual contaminants Buffer PB contains isopropanol and Gu. Cl Centrifuge PB buffer Nucleic acids PB + contaminants Buffer PE contains ethanol and Tris-Cl Centrifuge PE buffer Nucleic acids PE + contaminants (including residual Gu. Cl)
Elute purified DNA from the column Buffer EB should be added directly to the membrane for optimal DNA recovery and to avoid possible Et. OH contamination (from residual PE buffer) EB is 10 m. M Tris-Cl (p. H 8. 5). TE or d. H 2 O may also be used. Centrifuge EB buffer Nucleic acids EB + DNA
Manual alkaline lysis preparation of plasmid DNA • Organic extraction (optional) Mix thoroughly with an equal volume of organic solvent Aqueous Centrifuge e. g. phenol, chloroform, or phenol: chloroform Organic • Precipitate DNA with isopropanol (1: 1 volume) Supernatant 50% isopropanol + precipitated DNA Centrifuge Pellet • Wash pellet with 70% Et. OH (to remove salts) • Dissolve pellet with TE (or other aqueous solution)
Assessing your plasmid preparation 1. Quantify abundance (A 260) and purity (A 260/A 280) 2. Verify by restriction digestion 3. Run undigested plasmid to see if it is mostly supercoiled denatured
- Plasmid isolation principle
- Application of plasmid
- Recombinant dna
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