Captr.I etb Phepuntoe of amd DNA by Largest Boing Lysis Cold Spring harbor Molecular Cloning tory Prti A LABORATORY MANUAL ON THE WEB CHAPTER 1>PROTOCOL 5 0 printer friendly version Protocol 5 Preparation of Plasmid DNa by Large-scale Boiling Lysis asmd DNA is isdated from large-scale (500 ml) bacterial cutures by treatment with Triton X-100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA fom strains of E coli fhat express endonuclease A(endA" strains) MATERIALS A CAUTION: Please dick for information about appropriate handling of materials O RECIPE: Please dick for components of stock solutions, buffers, and reagents Buffers and solutions Antibiotic for plasmid selection △ chloramphenicol(34 mg/m thand Isopropanol O STE O STET 0正EcH80 Enzymes and Buffers 0 Lysozyme(10 mgml) Restriction endonucleases Media O Rich medium Additional Reagents tep 7 of this protocol requires the reagents listed in Chapter 1 Protocol 1 or Chapter 1, Protocol4 tep 20 of this protocol requires reagents listed in Chapter 1. Protoco 8, Chapter 1 Protocol 9, Chapter 1. Protocol 10. or Chanter 1. protoco 11 METHOD I. hoculate 30 ml of rich medium(LB, YT, or Temfic Broth) containing the appropriate anfibiofc either with a single colony of transformed bacteria or wth 0.1-1.0 ml of a small-scale liquid cuture grown from a single colony 2. haubate the cuture at the appropriate temperature with vigorous shaking (250 cycles/ minute in a rotary shaker)until the baceria reach the late log phase of growth (ie, an ODgm of approx. 0.6) 3. Inoculate 500 ml of LB, YT, or Terifc Broth(prewarmed to 37 C)containing the appropriate antibiotic in a2-liter fask with 25 ml of the late og-phase culture Incubate the alure for 2.5 hours at 37 C with vigorous shaking (250 cycles/minute on a rotary shaker) 4. Add 25 ml of 34 m ml chloramphenicol. The final concentration of chloramphenicol in the cuture should be 170 ug/mL. Incubate the culture for a further 12-16 hours at 37 C with vigorous shaking (250 cycles/minute an 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 clture by centrifugation at 2700g(4100 pm in a Soval GSA rotor for 15 minutes at 4'C. Discard the supernatant Stand the open centrifuge botile in an inverted position to alow al of the supematant 6. Resuspend the bacterial pellet in 200 ml of ice-cold STE Collect the bacterial cells by centrifugation as described in tep 5. Store the pellet af bacteria in the centrifuge bottle at-20C 7. Prepare plasmid DNA from the 1-2-mlaliquat of bacteria set aside in Step 5 by the minipreparation protocol(ether Protocol 1 or 4). Analyze the minipreparation plasmid DNA by digestion with the appropriate restriction enzyme(s)to ensure that the corred plasmid has been propagated in the large-scale culture. 8. Allow the frozen bacterial cell pellet from Step 6 to thaw for 510 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 mgiml lysozyme 10. Use a clamp to hold the Erlenmeyer fask over the open fame of a Bunsen bumer until the fiquid just starts to boil Shake the fask constantly during the heating procedure Il. Immediately immerse the bottom half of the flask ina large (2-iter beaker of boing 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 tbe(Beckman Sw41 or ts equivalent). Centrifuge the ysate at 150,000g (30,000 rpm in a Beckman Sw41Tirotar) for 30 minutes at4C I4. Transfer as much of the supernatant as possible to a new tube. Discard the viscous liquid remaining in the centrfuge 15.(optional) I the supernatant contains visble strings of genomic chromafin or fooalent precipitate of proteins, filter it through 4-ply gauze before proceeding 16. Measure the volume of the supematant. Transfer the supernatant, together with 0.6 volume of isopropanol, tb a fesh centrifuge tube(s). Store the tube(s)for 10 minutes at room temperature, after mixing the contents wel 17. Recover the precipitated nucleic acids by centrifugafion at 12, 000g (10,000 pm in a Sonvall SS-34 rotor) for 15 minutes at room temperature Salt may precipitate if centrifugation is carried out at 4( 18. Decant the supematant carefully, and invert the open tube(s) on a paper towel to allow the last drops of supematant to drain away. Rinse the pellet and the walls of the tube(s with 70% ethanol at room temperature. Drain off the ethand and use a Pasteur pipette attached to a vacuum line to remove any beads of iquid that adhere to the wals of the hube(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 pelet 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 centrifugafion in Csck-ethidium bromide grader Chapter 1. Protocol 10 and Chapter 1, Protoco 11). 21. Check the structure of the plasmid by restriction enzyme digestion followed by gel electophoresis REFERENCES I. Holmes D.S. and Quiley, 1981. A rapid boiling method for the preparafion of bacterial plasmids. Anal Bioc htprlrwwnoeaanirgaomlnebersprobalspprorumberslchprumbera1(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]