dOping habur Lazonby mss. Makala donn.Chapter Cold Spring harbor Molecular Cloning tory Prti A LABORATORY MANUAL ON THE WEB Protocols Chapter 1 Plasmids and Their Usefulness in Molecular Cloning Protocols Protocol 1: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation l Bioinform Plasmid DNA is solated from small-scale( 1-2m)bacterial cutures by teatment with alkali and SDS Protocol 2: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation Plasmid DNA is solated from intemediate-scale(20-50 ml)bactenal cultures by teatment with akali and SDs Protocol 3: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation Plasmid DNA is solated from large-scale(500 m) baderial cultures by treatment with alkali I Search Protocol 4: Preparation of Plasmid DNA by Smal-scale Boiling Lysis I Contact Us Plasmid DNA is solated from smal-scale( 1-2 m) bacterial cutures by treatment with Tnton X- 100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E cof that express endonuclease A (endA"strains) Protocol 5: Preparation of Plasmid DNA by Large-scale Boiling Lysis MEmbers Home Plasmid DNA is isolated from large-scale(500 ml)bateria atures by teatment with Titon X 100 and lysozyme, fallowed by heating. This method is not recommended for preparing plasmid DNA from strains of E coli that express endonuclease A(enda" strains). Protocol 6: Preparation of Plasmid DNA: Toothpick Minipreparation Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar neda with toothpicks Protocol 7: Preparation of Plasmid DNA by Lysis with SDS Large P15 kb), dosed aralar plasmids are prepared (albeit inefficienty and in small yeld) by ying bacteria with SDS Protocol 8: Purification of Plasmid DNA by Precipitation with Polyethylene Glycol Crude preparations of plasmid DNA are frst treated with ithium chloride and RNase (to ren RNA). The plasmid DNA is then preciptated in a solution containing polyethylene glyco and Protocol 9: Purification of Plasmid DNA by Chromatography The following table summarizes the salient features of many of the commercial resins that are curently avaiable for plasmid purification. Individual manufacturers supply detailed instructions which shoud be followed to the letter Protocol 10. Purification of Closed Circular DNA by Equilibrium Centrifugation in CsC. Ethidium Bromide Gradients: Continuous Gradients Solutions containing plasmid DNA are adjusted to a density of 1. 55 g/ml with sdid CsCl.The intercalating dye, ethidium bromide, which binds dilfterentialy to dosed cicular and linear NAs, is then added to a concentration of 200 ug ml. During centrifugation to equilibrium, the sed circular dna and linear dNAs form bands at different densities Protocol 11: Purification of Closed Circular DNA by Equilbrium Centrifugation in CSCh. Ethidium Bromide Gradients: Discontinuous Gradients A soluion containing plasmid DNA, saturating amounts of ethidium bromide, and CsCl (44% is layered between two solutions of lesser(35% wt CsC) and greater density (59%wlv Cl). During centriugation to equibrium, the dosed circular plasmid DNA and linear DNAs form bands at different densities Protocol 12 Removal of Ethidium Bromide from DNA by Extraction with Organic Ethidium bromide is removed from DNA by phase extraction wch organic solvents Protocol 13: Removal of Ethidium Bromide from DNA by lon-exchange Chromatography Ethidium bromide is removed from DNA by chromatography through a cation-exchange resin. Protocol 14: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation through Nacl Contamination of plasmid dNa by fragments of DNA and RNA is reduced to an acceptable level by centrifugation through 1 M sod um chloride. This method was devised by Brian Seed when he was a graduate student at anvard University Protocol 15: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Chromatography through SephacryIS-1000 Contamination of plasmid DNA by sma fragments of nudeic acid is reduced dramatically by ize-exdlusion chromatography through Sephacry/$-1000 Protocol 16: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Lithium Chloride High-molecular-weight RNA and proteins can be precipitated from preparaions of plasmid DNA by high concentrations of LiCl and removed by bow-speed centrifugation. Protocol 17: Directional Cloning into Plasmid Vectors Directional doning requires that the plasmid vector be cleaved wih two restriction enzymes that generate incompatible termini and that the fragment of DNA to be doned cares termini that are compatible with those of the doubly deaved vector Protocol 18: Attaching Adaptors to Protruding Termini Adaptors are short double-stranded synthetic oligonudeotides that carry an terma restriction d single-standed tails at one or both ends. Adaptors are used exchange restriction sites at the termini of inear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms Protocol 19. Blunt-ended Cloning into Plasmid Vectors 坤 wwnoeadarcbnig com/merberschapter sp?cha=1112012t母Cold Spring Harbor Laboratory Press - Molecular Cloning - Chapter 1 Chapter 1 Plasmids and Their Usefulness in Molecular Cloning Protocol 1: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with alkali and SDS. Protocol 2: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation Plasmid DNA is isolated from intermediate-scale (20-50 ml) bacterial cultures by treatment with alkali and SDS. Protocol 3: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with alkali and SDS. Protocol 4: Preparation of Plasmid DNA by Small-scale Boiling Lysis Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with Triton X- 100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). Protocol 5: Preparation of Plasmid DNA by Large-scale Boiling Lysis Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X- 100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). Protocol 6: Preparation of Plasmid DNA: Toothpick Minipreparation Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks. Protocol 7: Preparation of Plasmid DNA by Lysis with SDS Large (>15 kb), closed circular plasmids are prepared (albeit inefficiently and in small yield) by lysing bacteria with SDS. Protocol 8: Purification of Plasmid DNA by Precipitation with Polyethylene Glycol Crude preparations of plasmid DNA are first treated with lithium chloride and RNase (to remove RNA). The plasmid DNA is then precipitated in a solution containing polyethylene glycol and MgCl2. Protocol 9: Purification of Plasmid DNA by Chromatography The following table summarizes the salient features of many of the commercial resins that are currently available for plasmid purification. Individual manufacturers supply detailed instructions, which should be followed to the letter. Protocol 10: Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl￾Ethidium Bromide Gradients: Continuous Gradients Solutions containing plasmid DNA are adjusted to a density of 1.55 g/ml with solid CsCl. The intercalating dye, ethidium bromide, which binds differentially to closed circular and linear DNAs, is then added to a concentration of 200 µg/ml. During centrifugation to equilibrium, the closed circular DNA and linear DNAs form bands at different densities. Protocol 11: Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl￾Ethidium Bromide Gradients: Discontinuous Gradients A solution containing plasmid DNA, saturating amounts of ethidium bromide, and CsCl (44% w/v) is layered between two solutions of lesser (35% w/v CsCl) and greater density (59% w/v CsCl). During centrifugation to equilibrium, the closed circular plasmid DNA and linear DNAs form bands at different densities. Protocol 12: Removal of Ethidium Bromide from DNA by Extraction with Organic Solvents Ethidium bromide is removed from DNA by phase extraction with organic solvents. Protocol 13: Removal of Ethidium Bromide from DNA by Ion-exchange Chromatography Ethidium bromide is removed from DNA by chromatography through a cation-exchange resin. Protocol 14: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation through NaCl Contamination of plasmid DNA by fragments of DNA and RNA is reduced to an acceptable level by centrifugation through 1 M sodium chloride. This method was devised by Brian Seed when he was a graduate student at Harvard University. Protocol 15: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Chromatography through Sephacryl S-1000 Contamination of plasmid DNA by small fragments of nucleic acid is reduced dramatically by size-exclusion chromatography through Sephacryl S-1000. Protocol 16: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Lithium Chloride High-molecular-weight RNA and proteins can be precipitated from preparations of plasmid DNA by high concentrations of LiCl and removed by low-speed centrifugation. Protocol 17: Directional Cloning into Plasmid Vectors Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes that generate incompatible termini and that the fragment of DNA to be cloned carries termini that are compatible with those of the doubly cleaved vector. Protocol 18: Attaching Adaptors to Protruding Termini Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms. Protocol 19: Blunt-ended Cloning into Plasmid Vectors http://www.molecularcloning.com/members/chapter.jsp?chapter=112 (1 / 3) [2002-2-18 16:10:49]