Isolation of Plasmid DNA June 21, 2007 Leeward Community College.

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1 Isolation of Plasmid DNA June 21, 2007 Leeward Community College

2 E. coli Chromosome Plasmids DNA molecules separate from chromosomal DNA Self-replicating Electron micrograph of DNA from a lysed E. coli cell

3 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 substances Virulence plasmids: Causes the bacteria to act as a pathogen Plasmid Functional Categories Bacteria carrying a plasmid with the gene neomycin phosphotransferase are capable of surviving in the presence of the antibiotic kanamycin

4 Molecular Biology Applications for Plasmids I. Cloning of DNA fragments II. Protein Production + IPTG T7 promoter Gene of Interest Protein Induction Plasmid

5 Comparison of plasmid copy numbers in E. coli High copy DNA yield Likelihood of mutation Difficulty in cloning toxic genes Low copy

6 Gal4 DNA-binding Domain Bait Protein Gal4 Activation Domain Bait Vector Prey Vector Library Protein Molecular Biology Applications for Plasmids III. Yeast Two-Hybrid Assay: Tests for protein-protein interactions

7 Molecular Biology Applications for Plasmids IV. Agrobacterium-mediated Plant Transformation A means of performing plant genetic engineering

8 How to purify plasmid DNA using silica-based columns

9 Harvest cells by centrifugation Spin ~5,000 rcf Supernatant (clear)‏ Pelleted cells E. coli culture (cloudy)‏ Discard supernatant Residual media may interfere with downstream steps Resuspend cells in buffer Thoroughly resuspend cells, making sure that no clumps remain. P1 buffer contains: Tris-Cl (buffering agent)‏ EDTA (metal chelator)‏ RNase A (degrades RNA)‏

10 Dissolves membranes Binds to and denatures proteins 2. NaOH Denatures DNA Because plasmids are supercoiled, both DNA strands remain entangled after denaturation Lyse cells with SDS/NaOH solution Adding buffer P2 causes solution to become viscous 1. Sodium dodecyl sulfate

11 Neutralize NaOH with potassium acetate solution Mixing with buffer N3 causes a fluffy white precipitate to form. 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)‏ (H 2 O sol. = 10%) (H 2 O sol. < 0.02%)‏ 2. Guanidine hydrochloride (GuCl) Chaotropic salt; facilitates DNA binding to silica in later steps

12 Separate plasmid DNA from contaminants by centrifugation Supernatant contains: - Plasmid DNA - Soluble cellular constituents Pellet contains: - PDS - Lipids - Proteins - Chromosomal DNA

13 Add cleared lysate to column and centrifuge The high ionic strength and presence of chaotropic salt causes DNA to bind to the silica membrane, while other contaminants pass through the column Silica-gel membrane Centrifuge Flow through (discard)‏ Nucleic acids

14 Wash the silica membrane to remove residual contaminants Buffer PB contains isopropanol and GuCl Buffer PE contains ethanol and Tris-Cl Centrifuge PB + contaminants Nucleic acids PB buffer Centrifuge PE + contaminants (including residual GuCl)‏ Nucleic acids PE buffer

15 Elute purified DNA from the column EB is 10 mM Tris-Cl (pH 8.5). TE or dH 2 O may also be used. Centrifuge EB + DNA Nucleic acids EB buffer Buffer EB should be added directly to the membrane for optimal DNA recovery and to avoid possible EtOH contamination (from residual PE buffer)‏

16 Manual alkaline lysis preparation of plasmid DNA Organic extraction (optional)‏ Mix thoroughly with an equal volume of organic solvent e.g. phenol, chloroform, or phenol:chloroform Centrifuge Organic Aqueous Precipitate DNA with isopropanol (1:1 volume)‏ Supernatant Pellet Centrifuge 50% isopropanol + precipitated DNA Wash pellet with 70% EtOH (to remove salts)‏ Dissolve pellet with TE (or other aqueous solution)‏

17 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