《分子克隆》书籍PDF英文电子版（第三版）Cold Spring Harbor Laboratory Press《Molecular Cloning》

Chapter 1: Plasmids and Their Usefulness in Molecular Cloning Chapter 2: Bacteriophage and Its Vectors Chapter 3: Working with Bacteriophage M13 Vectors Chapter 4: Working with High-Capacity Vectors Chapter 5: Gel Electrophoresis of DNA and Pulsed-Field Agarose Chapter 6: Preparation and Analysis of Eukaryotic Genomic DNA Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells Chapter 8: In Vitro Amplification of DNA by the Polymerase Chain Reaction Chapter 9: Preparation of Radiolabeled DNA and RNA Probes Chapter 10: Working with Synthetic Oligonucleotide Probes Chapter 11: Preparation of cDNA Libraries and Gene Identification Chapter 12: DNA Sequencing Chapter 13: Mutagenesis Chapter 14: Screening Expression Libraries Chapter 15: Expression of Cloned Genes in Escherichia coli Chapter 16: Introducing Cloned Genes into Cultured Mammalian Cells Chapter 17: Analysis of Gene Expression in Cultured Mammalian Cells Chapter 18: Protein Interaction Technologies

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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 CsClEthidium 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 CsClEthidium 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]

Cold Spring Harbor Laboratory Press - Molecular Cloning - Chapter 1 Target DNA is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent E. coli. The maximum number of "correct" clones can generally be obtained from ligation reactions containing equimolar amounts of plasmid and target DNAs, with the total DNA concentration being <100 µg/ml. Blunt-end ligation catalyzed by bacteriophage T4 DNA ligase is suppressed by high concentrations (5 mM) of ATP and polyamines such as spermidine. Protocol 20: Dephosphorylation of Plasmid DNA During ligation in vitro, T4 DNA ligase will catalyze the formation of a phosphodiester bond between adjacent nucleotides only if one nucleotide carries a 5´-phosphate residue and the other carries a 3´-hydroxyl terminus. Recircularization of vector DNA can therefore be minimized by removing the 5´-phosphate residues from both termini of the linear, doublestranded plasmid DNA with alkaline phosphatase. Protocol 21: Addition of Synthetic Linkers to Blunt-ended DNA Linkers are small self-complementary pieces of synthetic DNA, usually 8-16 nucleotides in length, that anneal to form blunt-ended, double-stranded molecules containing a restriction site. Linkers are used to equip blunt-ended termini of DNA with restriction sites as an aid to cloning. Protocol 22: Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. Protocol 23: The Hanahan Method for Preparation and Transformation of Competent E. coli: High-efficiency Transformation This procedure generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/mg of superhelical plasmid DNA). IMPORTANT All steps in this protocol should be carried out aseptically. Protocol 24: The Inoue Method for Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells This protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/mg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C. Protocol 25: Preparation and Transformation of Competent E. coli Using Calcium Chloride This protocol, developed approx. 30 years ago, is used to prepare batches of competent bacteria that yield 5 x 106 to 2 x 107 transformed colonies/µg of supercoiled plasmid DNA. Protocol 26: Transformation of E. coli by Electroporation Electrocompetent bacteria are prepared by growing cultures to mid-log phase, washing the bacteria extensively at low temperature, and then resuspending them in a solution of low ionic strength containing glycerol. DNA is introduced during exposure of the bacteria to a short highvoltage electrical discharge. Protocol 27: Screening Bacterial Colonies Using X-gal and IPTG: -Complementation -complementation occurs when two inactive fragments of E. coli -galactosidase associate to form a functional enzyme. Many plasmid vectors carry a short segment of DNA containing the coding information for the first 146 amino acids of -galactosidase. Vectors of this type are used in host cells that express the carboxy-terminal portion of the enzyme. Although neither the host nor the plasmid-encoded fragments of -galactosidase are themselves active, they can associate to form an enzymatically active protein. Lac+ bacteria that result from - complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-gal. However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an amino-terminal fragment that is no longer capable of -complementation. Bacteria carrying recombinant plasmids therefore form white colonies. The development of this simple blue-white color test has greatly simplified the identification of recombinants constructed in plasmid vectors. Protocol 28: Screening Bacterial Colonies by Hybridization: Small Numbers This procedure, a variant of the Grunstein and Hogness (1979) method, is used to screen a small number of bacterial colonies (<200) that are dispersed over several agar plates and are to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar plate. After a period of growth, the colonies on the filter are lysed and processed for hybridization. The master plate is stored until the results of the screening procedure become available. Protocol 29: Screening Bacterial Colonies by Hybridization: Intermediate Numbers Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. This technique, a variant of the Grunstein and Hogness (1975) method, was developed at Cold Spring Harbor Laboratory in 1975. The procedure works best with 90-mm plates containing <2500 colonies. Protocol 30: Screening Bacterial Colonies by Hybridization: Large Numbers This procedure is used to plate, replicate, and subsequently screen large numbers of bacterial colonies (up to 2 x 104 colonies per 150-mm plate or 104 colonies per 90-mm plate). Protocol 31: Lysing Colonies and Binding of DNA to Filters In this protocol, based on the procedure of Grunstein and Hogness (1975), alkali is used to liberate DNA from bacterial colonies on nitrocellulose or nylon filters. The DNA is then fixed to the filter by UV-cross-linking or baking under vacuum. Protocol 32: Hybridization of Bacterial DNA on Filters This protocol describes procedures to hybridize DNA from transformed colonies immobilized on filters with radiolabeled probes and to recover from a master plate the corresponding colonies that hybridize specifically to the probe. The method is based on the procedure published by Grunstein and Hogness (1975). http://www.molecularcloning.com/members/chapter.jsp?chapter=112 (2 / 3) [2002-2-18 16:10:49]

Chapter:1 Protocol:1 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation CHAPTER 1 > PROTOCOL 1 printer friendly version 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. MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Alkaline lysis solution I Alkaline lysis solution II Alkaline lysis solution III Antibiotic for plasmid selection Ethanol Phenol:chloroform (1:1, v/v) STE TE (pH 8.0) containing 20 µg/ml RNase A Media Rich medium METHOD 1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking. 2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C. 3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible. 4. Resuspend the bacterial pellet in 100 µl of ice-cold Alkaline lysis solution I by vigorous vortexing. 5. Add 200 µl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice. 6. Add 150 µl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes. 7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge. Transfer the supernatant to a fresh tube. 8. (Optional) Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube. 9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature. 10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge. 11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube. 12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge. 13. Remove all of the supernatant by gentle aspiration as described in Step 3.Take care with this step, as the pellet sometimes does not adhere tightly to the tube. 14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes). 15. Dissolve the nucleic acids in 50 µl of TE (pH 8.0) containing 20 µg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds. Store the DNA solution at -20°C. REFERENCES 1. Birnboim H.C. and Doly J. 1979. A rapid alkaline procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 2. Ish-Horowicz D. and Burke J.F. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res. 9:2989-2998. printer friendly version Buy The Book | Our Vision | Take The Tour | Newsletter | Search CSHL Press Home | Contact | Members Home | CSHL Home Copyright © 2000. Cold Spring Harbor Laboratory Press. http://www.molecularcloning.com/members/protocol.jsp?pronumber=1&chpnumber=1 [2002-2-18 16:11:00]

Chapter:1 Protocol:2 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation CHAPTER 1 > PROTOCOL 2 printer friendly version 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. MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Alkaline lysis solution I For preparations of plasmid DNA that are to be subjected to further purification by chromatography (please see Chapter 1, Protocol 9 ), sterile Alkaline lysis solution I may be supplemented just before use with the appropriate volume of 20 mg/ml DNase-free RNase A (pancreatic RNase) to give a final concentration of 100 µg/ml. Alkaline lysis solution II Alkaline lysis solution III Antibiotic for plasmid selection Ethanol Isopropanol Phenol:chloroform (1:1, v/v) STE TE (pH 8.0) containing 20 µg/ml RNase A Media Rich medium METHOD 1. Inoculate 10 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking. 2. Transfer the culture into a 15-ml tube and recover the bacteria by centrifugation at 2000g (4000 rpm in a Sorvall SS-34 rotor) for 10 minutes at 4°C. 3. Remove the medium by gentle aspiration, leaving the bacterial pellet as dry as possible. 4. Resuspend the bacterial pellet in 200 µl of ice-cold Alkaline lysis solution I by vigorous vortexing, and transfer the suspension to a microfuge tube. 5. Add 400 µl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice. 6. Add 300 µl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes. 7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge. Transfer 600 µl of the supernatant to a fresh tube. 8. Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube. 9. Precipitate nucleic acids from the supernatant by adding 600 µl of isopropanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature. 10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at room temperature in a microfuge. 11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Remove any drops of fluid adhering to the walls of the tube. 12. Add 1 ml of 70% ethanol to the pellet and recover the DNA by centrifugation at maximum speed for 2 minutes at room temperature in a microfuge. 13. Remove all of the supernatant by gentle aspiration as described in Step 3. 14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (2-5 minutes). 15. Dissolve the nucleic acids in 100 µl of TE (pH 8.0) containing 20 µg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds and store at -20°C. REFERENCES 1. Birnboim H.C. and Doly J. 1979. A rapid alkaline procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 2. Ish-Horowicz D. and Burke J.F. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res. 9:2989-2998. printer friendly version Buy The Book | Our Vision | Take The Tour | Newsletter | Search CSHL Press Home | Contact | Members Home | CSHL Home Copyright © 2000. Cold Spring Harbor Laboratory Press. http://www.molecularcloning.com/members/protocol.jsp?pronumber=2&chpnumber=1 [2002-2-18 16:11:10]

Chapter:1 Protocol:3 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation CHAPTER 1 > PROTOCOL 3 printer friendly version 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. MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Alkaline lysis solution I For preparations of plasmid DNA that are to be subjected to further purification by chromatography (please see Chapter 1, Protocol 9 ), sterile Alkaline lysis solution I may be supplemented just before use with the appropriate volume of 20 mg/ml DNase-free RNase A (pancreatic RNase) to give a final concentration of 100 µg/ml Alkaline lysis solution II Alkaline lysis solution III Antibiotic for plasmid selection Chloramphenicol (34 mg/ml) Ethanol Isopropanol STE TE (pH 8.0) Enzymes and Buffers Lysozyme (10 mg/ml) Restriction endonucleases Media Rich medium Additional Reagents Steps 8 and 19 of this protocol require reagents listed in Chapter 5, Protocol 1 . Step 18 of this protocol requires reagents listed in Chapter 1, Protocol 8 , Chapter 1, Protocol 9 , Chapter 1, Protocol 10 , or Chapter 1, Protocol 11 . METHOD 1. Inoculate 30 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic either with a single colony of transformed bacteria or with 0.1-1.0 ml of a small-scale liquid culture grown from a single colony. 2. Incubate the culture at the appropriate temperature with vigorous shaking until the bacteria reach late log phase (OD600 = approx. 0.6). 3. Inoculate 500 ml of LB, YT, or Terrific Broth medium (prewarmed to 37°C) containing the appropriate antibiotic in a 2- liter flask with 25 ml of the late-log-phase culture. Incubate the culture for approx. 2.5 hours at 37°C with vigorous shaking (300 cycles/minute on a rotary shaker). 4. For relaxed plasmids with low or moderate copy numbers, add 2.5 ml of 34 mg/ml chloramphenicol solution. The final concentration of chloramphenicol in the culture should be 170 µg/ml. For high-copy-number plasmids, do not add chloramphenicol. 5. Incubate the culture for a further 12-16 hours at 37°C with vigorous shaking (300 cycles/minute on a rotary shaker). 6. Remove an aliquot (1-2 ml) of the bacterial culture to a fresh microfuge tube and store it at 4°C. Harvest the remainder of the bacterial cells from the 500-ml culture by centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 15 minutes at 4°C. Discard the supernatant. Stand the open centrifuge bottle in an inverted position. 7. Resuspend the bacterial pellet in 200 ml of ice-cold STE. Collect the bacterial cells by centrifugation as described in Step 6. Store the pellet of bacteria in the centrifuge bottle at -20°C. 8. Use one of the methods described in Chapter 1, Protocol 1 or Chapter 1, Protocol 4 to prepare plasmid DNA from the 1- 2-ml aliquot of bacterial culture set aside in Step 6. Analyze the minipreparation plasmid DNA by digestion with the appropriate restriction enzyme(s) and agarose gel electrophoresis to ensure that the correct plasmid has been propagated in the large-scale culture. 9. Allow the frozen bacterial cell pellet from Step 7 to thaw at room temperature for 5-10 minutes. Resuspend the pellet in 18 ml (10 ml) of Alkaline lysis solution I. 10. Add 2 ml (1 ml) of a freshly prepared solution of 10 mg/ml lysozyme. 11. Add 40 ml (20 ml) of freshly prepared Alkaline lysis solution II. Close the top of the centrifuge bottle and mix the contents thoroughly by gently inverting the bottle several times. Incubate the bottle for 5-10 minutes at room temperature. 12. Add 20 ml (15 ml) of ice-cold Alkaline lysis solution III. Close the top of the centrifuge bottle and mix the contents gently but well by swirling the bottle several times (there should no longer be two distinguishable liquid phases). Place the bottle on ice for 10 minutes. 13. Centrifuge the bacterial lysate at 20,000g (11,000 rpm in a Sorvall GSA rotor) for 30 minutes at 4°C in a mediumspeed centrifuge. Allow the rotor to stop without braking. At the end of the centrifugation step, decant the clear supernatant into a graduated cylinder. Discard the pellet remaining in the centrifuge bottle. 14. Measure the volume of the supernatant. Transfer the supernatant together with 0.6 volume of isopropanol to a fresh centrifuge bottle. Mix the contents well and store the bottle for 10 minutes at room temperature. 15. Recover the precipitated nucleic acids by centrifugation at 12,000g (8000 rpm in a Sorvall GSA rotor) for 15 minutes at room temperature. 16. Decant the supernatant carefully, and invert the open bottle on a paper towel to allow the last drops of supernatant to drain away. Rinse the pellet and the walls of the bottle with 70% ethanol at room temperature. Drain off the ethanol, and use a Pasteur pipette attached to a vacuum line to remove any beads of liquid that adhere to the walls of the bottle. Place the inverted, open bottle on a pad of paper towels for a few minutes at room temperature. 17. Dissolve the damp pellet of nucleic acid in 3 ml of TE (pH 8.0). 18. Purify the crude plasmid DNA either by column chromatography ( Chapter 1, Protocol 9 ), precipitation with polyethylene glycol ( Chapter 1, Protocol 8 ), or equilibrium centrifugation in CsCl-ethidium bromide gradients ( Chapter 1, Protocol 10 and Chapter 1, Protocol 11 ). 19. Check the structure of the plasmid by restriction enzyme digestion followed by gel electrophoresis. REFERENCES http://www.molecularcloning.com/members/protocol.jsp?pronumber=3&chpnumber=1 (1 / 2) [2002-2-18 16:11:19]

Chapter:1 Protocol:4 Preparation of Plasmid DNA by Small-scale Boiling Lysis CHAPTER 1 > PROTOCOL 4 printer friendly version 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). MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Antibiotic for plasmid selection Ethanol Isopropanol Sodium acetate (3.0 M, pH 5.2) STET TE (pH 8.0) containing 20 µg/ml RNase A Enzymes and Buffers Lysozyme (10 mg/ml) Media Rich medium METHOD 1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking. 2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge the tube at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the culture at 4°C. 3. Remove the medium by gentle aspiration, leaving the bacterial pellet as dry as possible. 4. Resuspend the bacterial pellet in 350 µl of STET. 5. Add 25 µl of a freshly prepared solution of lysozyme. Close the top of the tube and mix the contents by gently vortexing for 3 seconds. 6. Place the tube in a boiling water bath for exactly 40 seconds. 7. Centrifuge the bacterial lysate at maximum speed for 15 minutes at room temperature in a microfuge. Pour the supernatant into a fresh microfuge tube. 8. Precipitate the nucleic acids from the supernatant by adding 40 µl of 2.5 M sodium acetate (pH 5.2) and 420 µl of isopropanol. Mix the solution by vortexing, and then allow the mixture to stand for 5 minutes at room temperature. 9. Recover the precipitated nucleic acids by centrifugation at maximum speed for 10 minutes at 4°C in a microfuge. 10. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube. 11. Rinse the pellet of nucleic acid with 1 ml of 70% ethanol at 4°C. Remove all of the supernatant by gentle aspiration as described in Step 3. 12. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (2-5 minutes). 13. Dissolve the nucleic acids in 50 µl of TE (pH 8.0) containing DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a brief period. Store the DNA at -20°C. REFERENCES 1. Holmes D.S. and Quigley M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193-197. printer friendly version Buy The Book | Our Vision | Take The Tour | Newsletter | Search CSHL Press Home | Contact | Members Home | CSHL Home Copyright © 2000. Cold Spring Harbor Laboratory Press. http://www.molecularcloning.com/members/protocol.jsp?pronumber=4&chpnumber=1 [2002-2-18 16:11:35]

Chapter:1 Protocol:5 Preparation of Plasmid DNA by Large-scale Boiling Lysis CHAPTER 1 > PROTOCOL 5 printer friendly version 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). MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Antibiotic for plasmid selection Chloramphenicol (34 mg/ml) Ethanol Isopropanol STE STET TE (pH 8.0) Enzymes and Buffers Lysozyme (10 mg/ml) Restriction endonucleases Media Rich medium Additional Reagents Step 7 of this protocol requires the reagents listed in Chapter 1, Protocol 1 or Chapter 1, Protocol 4 . Step 20 of this protocol requires reagents listed in Chapter 1, Protocol 8 , Chapter 1, Protocol 9 , Chapter 1, Protocol 10 , or Chapter 1, Protocol 11 . METHOD 1. Inoculate 30 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic either with a single colony of transformed bacteria or with 0.1-1.0 ml of a small-scale liquid culture grown from a single colony. 2. Incubate the culture at the appropriate temperature with vigorous shaking (250 cycles/ minute in a rotary shaker) until the bacteria reach the late log phase of growth (i.e., an OD600 of approx. 0.6). 3. Inoculate 500 ml of LB, YT, or Terrific Broth (prewarmed to 37°C) containing the appropriate antibiotic in a 2-liter flask with 25 ml of the late-log-phase culture. Incubate the culture for 2.5 hours at 37°C with vigorous shaking (250 cycles/minute on a rotary shaker). 4. Add 2.5 ml of 34 mg/ml chloramphenicol. The final concentration of chloramphenicol in the culture should be 170 µg/ml. Incubate the culture for a further 12-16 hours at 37°C with vigorous shaking (250 cycles/minute on a rotary shaker). 5. Remove an aliquot (1-2 ml) of the bacterial culture to a fresh microfuge tube and store at 4°C. Harvest the remainder of the bacterial cells from the 500-ml culture by centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 15 minutes at 4°C. Discard the supernatant. Stand the open centrifuge bottle in an inverted position to allow all of the supernatant to drain away. 6. Resuspend the bacterial pellet in 200 ml of ice-cold STE. Collect the bacterial cells by centrifugation as described in Step 5. Store the pellet of bacteria in the centrifuge bottle at -20°C. 7. Prepare plasmid DNA from the 1-2-ml aliquot of bacteria set aside in Step 5 by the minipreparation protocol (either Protocol 1 or 4). Analyze the minipreparation plasmid DNA by digestion with the appropriate restriction enzyme(s) to ensure that the correct plasmid has been propagated in the large-scale culture. 8. Allow the frozen bacterial cell pellet from Step 6 to thaw for 5-10 minutes at room temperature. Resuspend the pellet in 10 ml of ice-cold STET. Transfer the suspension to a 50-ml Erlenmeyer flask. 9. Add 1 ml of a freshly prepared solution of 10 mg/ml lysozyme. 10. Use a clamp to hold the Erlenmeyer flask over the open flame of a Bunsen burner until the liquid just starts to boil. Shake the flask constantly during the heating procedure. 11. Immediately immerse the bottom half of the flask in a large (2-liter) beaker of boiling water. Hold the flask in the boiling water for exactly 40 seconds. 12. Cool the flask in ice-cold water for 5 minutes. 13. Transfer the viscous contents of the flask to an ultracentrifuge tube (Beckman SW41 or its equivalent). Centrifuge the lysate at 150,000g (30,000 rpm in a Beckman SW41Ti rotor) for 30 minutes at 4°C. 14. Transfer as much of the supernatant as possible to a new tube. Discard the viscous liquid remaining in the centrifuge tube. 15. (Optional) If the supernatant contains visible strings of genomic chromatin or flocculent precipitate of proteins, filter it through 4-ply gauze before proceeding. 16. Measure the volume of the supernatant. Transfer the supernatant, together with 0.6 volume of isopropanol, to a fresh centrifuge tube(s). Store the tube(s) for 10 minutes at room temperature, after mixing the contents well. 17. Recover the precipitated nucleic acids by centrifugation at 12,000g (10,000 rpm in a Sorvall SS-34 rotor) for 15 minutes at room temperature. Salt may precipitate if centrifugation is carried out at 4°C. 18. Decant the supernatant carefully, and invert the open tube(s) on a paper towel to allow the last drops of supernatant to drain away. Rinse the pellet and the walls of the tube(s) with 70% ethanol at room temperature. Drain off the ethanol, and use a Pasteur pipette attached to a vacuum line to remove any beads of liquid that adhere to the walls of the tube(s). Place the inverted, open tube(s) on a pad of paper towels for a few minutes at room temperature. The pellet should still be damp. 19. Dissolve the pellet of nucleic acid in 3 ml of TE (pH 8.0). 20. Purify the crude plasmid DNA either by chromatography on commercial resins (Chapter 1, Protocol 9), precipitation with polyethylene glycol ( Chapter 1, Protocol 8), or equilibrium centrifugation in CsCl-ethidium bromide gradients ( Chapter 1, Protocol 10 and Chapter 1, Protocol 11 ). 21. Check the structure of the plasmid by restriction enzyme digestion followed by gel electrophoresis. REFERENCES 1. Holmes D.S. and Quigley M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. http://www.molecularcloning.com/members/protocol.jsp?pronumber=5&chpnumber=1 (1 / 2) [2002-2-18 16:11:46]

Chapter:1 Protocol:6 Preparation of Plasmid DNA: Toothpick Minipreparation CHAPTER 1 > PROTOCOL 6 printer friendly version 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. MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Antibiotic for plasmid selection Bromophenol blue solution (0.4% w/v) Cresol red solution (10 mM) EDTA (0.5 M, pH 8.0) Ethidium bromide (10 mg/ml) SYBR Gold KCl (4 M) NSS solution Media Rich broth Rich broth agar plates Additional Reagents Step 12 of this protocol requires the reagents listed in Chapter 8, Protocol 1 . Step 14 of this protocol requires the reagents listed in Chapter 1, Protocol 1 or Chapter 1, Protocol 4 . METHOD 1. Grow bacterial colonies, transformed with recombinant plasmid, on rich agar medium (LB, YT, or SOB) containing the appropriate antibiotic until they are approx. 2-3 mm in diameter (approx. 18-24 hours at 37°C for most bacterial strains). 2. Use a sterile toothpick or disposable loop to transfer a small segment of a bacterial colony to a streak or patch on a master agar plate containing the appropriate antibiotic. Transfer the remainder of the colony to a numbered microfuge tube containing 50 µl of sterile 10 mM EDTA (pH 8.0). 3. Repeat Step 2 until the desired number of colonies has been harvested. 4. Incubate the master plate for several hours at 37°C and then store it at 4°C until the results of the gel electrophoresis (Step 11 of this protocol) are available. Colonies containing plasmids of the desired size can then be recovered from the master plate. 5. While the master plate is incubating, process the bacterial suspensions as follows: To each microfuge tube in turn, add 50 µl of a freshly made solution of NSS. Close the top of the tubes and then mix their contents by vortexing for 30 seconds. 6. Transfer the tubes to a 70°C water bath. Incubate the tubes for 5 minutes and then allow them to cool to room temperature. 7. To each tube, add 1.5 µl of a solution of 4 M KCl. Vortex the tubes for 30 seconds. 8. Incubate the tubes for 5 minutes on ice. 9. Remove bacterial debris by centrifugation at maximum speed for 3 minutes at 4°C in a microfuge. 10. Transfer each of the supernatants in turn to fresh microfuge tubes. Add to each tube 0.5 µl of a solution containing 0.4% bromophenol blue if the samples are to be analyzed only by agarose gel electrophoresis or 2 µl of 10 mM cresol red if the samples are to be analyzed both by PCR and by agarose gel electrophoresis. Load 50 µl of the supernatant into a slot (5 mm in length x 2.5 mm in width) cast in a 0.7% agarose gel (5 mm thick). 11. After the bromophenol blue dye has migrated two-thirds to three-fourths the length of the gel, or the cresol red dye about one-half the length of the gel, stain the gel by soaking it for 30-45 minutes in a DNA-staining solution at room temperature. Examine and photograph the gel under UV illumination. 12. If cresol red has been used at Step 10, analyze the supernatants by performing PCR as described in Chapter 8, Protocol 1 , using the remainder of each sample as a template. 13. Prepare small-scale cultures of the putative recombinant clones by inoculating 2 ml of liquid medium (LB, YT, or SOB) containing the appropriate antibiotic with bacteria growing on the master plate. 14. Use the small-scale bacterial cultures to generate minipreparations (please see Chapter 1, Protocol 1 or Chapter 1, Protocol 4 ) of the putative recombinant plasmids. Analyze the plasmid DNAs by digestion with restriction enzymes and agarose gel electrophoresis to confirm that they have the desired size and structure. REFERENCES 1. Barnes W.M. 1977. Plasmid detection and sizing in single colony lysates. Science 195:393-394. printer friendly version Buy The Book | Our Vision | Take The Tour | Newsletter | Search CSHL Press Home | Contact | Members Home | CSHL Home Copyright © 2000. Cold Spring Harbor Laboratory Press. http://www.molecularcloning.com/members/protocol.jsp?pronumber=6&chpnumber=1 [2002-2-18 16:11:58]

Chapter:1 Protocol:7 Preparation of Plasmid DNA by Lysis with SDS CHAPTER 1 > PROTOCOL 7 printer friendly version 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. MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Antibiotic for plasmid selection Chloramphenicol (34 mg/ml) Chloroform EDTA (0.5 M, pH 8.0) Ethanol NaCl (5 M) Phenol:chloroform (1:1, v/v) SDS (10% w/v) STE, ice cold TE (pH 8.0) Tris-sucrose Enzymes and Buffers Lysozyme (10 mg/ml) Restriction endonucleases Media Rich medium Additional Reagents Step 8 of this protocol requires the reagents listed in Chapter 1, Protocol 1 or Chapter 1, Protocol 4 . Step 21 of this protocol requires the reagents listed in Chapter 1, Protocol 9 or Chapter 1, Protocol 10 . METHOD 1. Inoculate 30 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single transformed bacterial colony or with 0.1-1.0 ml of a late-log-phase culture grown from a single transformed colony. 2. Incubate the culture with vigorous shaking until the bacteria enters the late log phase of growth (i.e., an OD600 of approx. 0.6). 3. Inoculate 500 ml of LB, YT, or Terrific Broth (prewarmed to 37°C) containing the appropriate antibiotic in a 2-liter flask with 25 ml of the late-log-phase culture. Incubate the culture for approx. 2.5 hours at 37°C with vigorous shaking (250 cycles/minute on a rotary shaker). 4. For relaxed plasmids with low or moderate copy numbers, add 2.5 ml of 34 mg/ml chloramphenicol. The final concentration of chloramphenicol in the culture should be 170 µg/ml. For high-copy-number plasmids, do not add chloramphenicol. 5. Incubate the culture for a further 12-16 hours at 37°C with vigorous shaking (250 cycles/minute on a rotary shaker) 6. Remove an aliquot (1-2 ml) of the bacterial culture to a fresh microfuge tube and store it at 4°C. Harvest the remainder of the bacterial cells from the 500-ml culture by centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 15 minutes at 4°C. Discard the supernatant. Stand the open centrifuge bottle in an inverted position. 7. Resuspend the bacterial pellet in 200 ml of ice-cold STE. Collect the bacterial cells by centrifugation as described in Step 6. Store the pellet of bacteria in the centrifuge bottle at -20°C. 8. Use one of the methods described in Chapter 1, Protocol 1 or Chapter 1, Protocol 4 to prepare plasmid DNA from the 1- 2-ml aliquot of bacterial culture set aside in Step 6. Analyze the minipreparation plasmid DNA by digestion with the appropriate restriction enzyme(s) and agarose gel electrophoresis to ensure that the correct plasmid has been propagated in the large-scale culture. 9. Allow the frozen bacterial cell pellet from Step 7 to thaw at room temperature for 5-10 minutes. Resuspend the pellet in 10 ml of ice-cold Tris-sucrose solution. Transfer the suspension to a 30-ml plastic screw-cap tube. 10. Add 2 ml of a freshly prepared lysozyme solution (10 mg/ml) followed by 8 ml of 0.25 M EDTA (pH 8.0). 11. Mix the suspension by gently inverting the tube several times. Store the tube on ice for 10 minutes. 12. Add 4 ml of 10% SDS. Immediately mix the contents of the tube with a glass rod so as to disperse the solution of SDS evenly throughout the bacterial suspension. Be as gentle as possible to minimize shearing of the liberated chromosomal DNA. 13. As soon as mixing is completed, add 6 ml of 5 M NaCl (final concentration = 1 M). Use a glass rod to mix the contents of the tube gently but thoroughly. Place the tube on ice for at least 1 hour. 14. Remove high-molecular-weight DNA and bacterial debris by centrifugation at 71,000g (30,000 rpm in a Beckman Type 50 rotor) for 30 minutes at 4°C. Carefully transfer the supernatant to a 50-ml disposable plastic centrifuge tube. Discard the pellet. 15. Extract the supernatant once with phenol:chloroform and once with chloroform. 16. Transfer the aqueous phase to a 250-ml centrifuge bottle. Add 2 volumes (approx. 60 ml) of ethanol at room temperature. Mix the solution well. Store the solution for 1-2 hours at room temperature. 17. Recover the nucleic acids by centrifugation at 5000g (5500 rpm in a Sorvall GSA rotor or 5100 rpm in a Sorvall HS4 swing-out rotor) for 20 minutes at 4°C. 18. Discard the supernatant. Wash the pellet and sides of the centrifuge tube with 70% ethanol at room temperature and then centrifuge as in Step 17. 19. Discard as much of the ethanol as possible, and then invert the centrifuge bottle on a pad of paper towels to allow the last of the ethanol to drain away. Use a vacuum aspirator to remove droplets of ethanol from the walls of the centrifuge bottle. Stand the bottle in an inverted position until no trace of ethanol is visible. At this stage, the pellet should still be damp. 20. Dissolve the damp pellet of nucleic acid in 3 ml of TE (pH 8.0). 21. Purify the crude plasmid DNA either by chromatography on commercial resins (please see Chapter 1, Protocol 9 ) or isopycnic centrifugation in CsCl-ethidium bromide gradients (please see Chapter 1, Protocol 10 and Chapter 1, Protocol 11 ). 22. Check the structure of the plasmid by restriction enzyme digestion followed by gel electrophoresis. REFERENCES http://www.molecularcloning.com/members/protocol.jsp?pronumber=7&chpnumber=1 (1 / 2) [2002-2-18 16:12:11]

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