Chaper I Petot.3 Pepuntom of smd DNA by Alane Lygs wth SDs: Mapmeparan Cold Spring harbor Molecular Cloning tory Prti A LABORATORY MANUAL ON THE WEB CHAPTER 1>PROTOCOL3 0 printer friendly version Protocol 3 Preparation of Plasmid DNa by Alkaline Lysis with SDS: Maxipreparation Plasmid DNA is isdated from large-scale (500 ml) bacterial cutures by treatment with alkali and SDS MATERIALS A CAUTON: Please dick for information about appropriate handing of materias. O RECIPE: Please dik for components of stock solutions, buffers, and reagents Buffers and solutions O Alkaine lysis solufion I Far preparations of plasmid DNA that are to be subjected to further pumica ion by chromatography (please see Chapter 1, Protocol 9), sterile Alkaline lais solution I may be supplemented just before use with the appmpriat volume df 20 mgm/ DNase-free RNase A /pancreatic RNase)to give a final concentration of 100/ml O Alkaline lysis solufion ll O Alkaline lysis solufion Il Antibiotic for plasmid selection A Chloramphenicol (34 mg/)y Isopropanol 0 0正EpH80 Enzymes and Buffers 0 Lysozyme(10 mgml) estriction endonucleases Vedia 0 Rich medium Additional Reagents Steps 8 and 19 of this protocol require reagents isted in Chapter 5, Protocol 1 tep 18 of this protocol requires reagents isted in Chapter 1 Protocol 8. Chapter 1 Protocol 9, Chapter 1. Protocol 10. or Chanter 1. Protoco 11 METHOD I. inoculate 30 ml of rich medium (LB, YT, or Temfc Broth) containing the appropriate anfibiofc either with a single colony of transformed bacteria or with 0.1-1.0 ml of a smal-scale liquid cuture grown from a single colony 2. Incubate the culture at the appropriate temperature with vigorous shaking until the bacteria reach late lg phase(0D zo a006 3. Inoculate 500 ml of LB, YT, ar Terifc Broth medum(prewarmed to 37 C) containing the appropriate antibiotic in a 2 iter fask with 25 ml of the latelog-phase cuture. Incubate the culture for approx. 25 hours at 3T C with vigorous shaking (300 cydeshminute on a rotary shaker) 4. For relaxed plasmids with bow or moderate copy numbers, add 2.5 ml of 34 mg/ml chloramphenicol solution. The final concentration of chloramphenicol in the ature should be 170 glm For high-oop -number plasmids, do not add chloramphenicol 5. haubate the cuture for a further 12-16 hours at 37 C with vigorous shaking(300 cycles/minute on a rotay shaker) 6. Remove an aliquot 1-2 ml) of the bacterial allure to a fresh miarofuge tube and store it at 4'C. Harvest the remainder of the bacterial cels from the 500-ml culture by centrifugation at 2700g(4100 pm na Sorvall GSA rotor) for 15 minutes at4C Discard the supematant Stand the open centrifuge bottle in an inverted positi Resuspend the bacterial pellet in 200 m of ice-cold STE Collect the bacterial cels by centrifugation as desorbed in Step 6. Store the pellet f bacteria in the centrifuge botle at-20C 8. Use one of the methods described in Chapter 1. Protocol 1 or Chanter 1. Prot ocol 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 Aka ine lysis solufion L 10. Add 2 ml (1 m) of a freshly prepared solution of 10 mgiml lysozyme Il. Add 40 ml (20 ml) of freshly prepared Alkaline lysis solution Il. Close the top of the centrifuge bottle and mix the contents thoroughly by gently inverting the botle several imes Incubate the bottle for 5-10 minutes at room 12. Add 20 ml(15 ml) of ice-cold Alkaline lysis solufion ll. Close the top of the centrifuge bottle and mix the cont ents gently bu well by swirling the bottle several imes (there should no longer be two distinguishable liqud phases). Pace the botle on ice for 10 minues 13. Centrifuge the bacterial lysate at 220,00g(11,000 pm na Sorvall GSA rotor) for 30 minutes at 4 C in a medium- speed centrifuge. Alow the rotor to stop without braking. At the end of the centrifugation step, decant the clear supernatant into a graduated cyinder. Discard the pellet remaining in the centrifuge bottle I4. Measure the volume of the supematant. Transfer the supernatant together with 0.6 volume of isopropanol to a fresh centrifuge botte. Mix the contents well and store the bottle for 10 minutes at room temperal 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 supematant carefully, and invert the open bottle on a paper towel to alow the last drops of supematant to dran away. Rinse the pellet and the walls of the botle 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 btle. Place the inverted, open botte on a pad of paper towels for a few minutes at room temperature. 17. Dissove the damp pellet of nudeic acid in 3 ml of TE (H 8.0) 18. Purify the crude plasmid DNA either by colum chromatography ( Chapter 1 Protocol 9), precipitation with thylene gycol ( Chapter 1. Protocol 8), or equilibrum centrifugation in CsCHethidium 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 htp hwwnoeaarnigcommembersproblipsp?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 medium￾speed 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]