Molecular Cloning CHAPTER1>PROTOCOL Protocol 3 Proparation of Plasmid DNA by Aikaline Lyswith DS:Maxipreparation O TE (PH 8.0) Media Rich medu 8 and 19 of this p METHOD log phase (OO .0.6 300 gi-cop 27 e pe d in the 13. the er b col 11 REFERENCES
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]
Molecular Cloning L A BO R A T OR Y CHAPTER1>PROTOCOL printeriendve Protocol4 etale (3.0 M.pH 5.Z) O STET TE (pH 8.0)oo Rich medum METHOD r30 seconds4"℃nf ion of t 25 be in a boil rature in a micrctuge.Pour the atant by gentle aspiraon a 13. REFERENCES ation of ba printor friondly vorsion 0ee
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]
Molecular Cloning CHAPTER1>PROTOCOLS Protocol5 Proparation of Plasmid DNA by on Lysis MATERIALS 合etwa O TE (GH8.0) Media METHOD oloe-10mcleinl cydes/minute in a rotary shaker)unt DNA by om temperalure.Resuspend the pelet in s to boa lquld remaining in the contrituge 15. 16.1 ol,to a fresh 17. () the c REFERENCES
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]
@片Molecular Cloning L A B O R A T O RY Protocol 6 Preparation of Plasr id DN thpick Minipreparation 8e METHOD ning the the tubes to a 7C water bath.Incubale the tubes for 5 minutes and then allow them to cool to room of4 M KCL Vortex the tubes for 30 seconds r3 R9 ose gel (5 mm s by performing PCR as de d in ChapterB. o in 14.Use the REFERENCES 1.Barnea W.M.1977.Platm Press
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]
Molecular Cloning CHAPTER1>PROTOCOL7 Protocol7 Proparation of Plasmid DNA by Lysis with SDS EDTA (O5 M.PH8.0) NaCI(M) (10%wV) (, Siep 21 of this p METHOD the re by ce nt270 gation as described in 1.Pro 0m0r w-cap (PH 8.0 0 ut the 13 u-mlentlom and sides of the tube with 70%ethanol at room lemperatu ( 24 of the d by gelele REFERENCES
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]