Chapter I Phet 7 Pepuntm f amd DNA by Lysis aith SDS Cold Spring harbor Molecular Cloning tory Prti A LABORATORY MANUAL ON THE WEB CHAPTER 1>PROTOCOL 7 0 printer friendly version Protocol 7 Preparation of Plasmid DNa by Lysis with SDS Large(P15 kb), dosed circular plasmids are prepared (a bet neficienfy and in small yeld) by lysing bactenia with 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 Antibiotic for plasmid selection △ chloramphenicol(34 mg/m A Chloroform O EDTA(0.5M, PH 8. 0) O NaCl (5 M) △ Phenotchoroform A0S0s(10% O STE ice cold O TE (pH 8.0) 0 Tris-sucrose nzymes and Buffers 0 Lysozyme(10 mgm) Restriction endonucleases 0 Rich medim Additional Reagents Step 8 of this protocol requires the reagents listed in Chapter 1, Protocol 1 or Chapter 1. Protocol4 tep 21 of this protocol requires the reagents isted in Chapter 1, Protoca g or Chapter 1, Protocol 10 METHOD I. hoculate 30 ml of rich medium(LB, YT, or Temfic Broth) containing the appropriate antibiotic with a single transformed bactenia colony or with 0. 1.1.0 ml of a late- og -phase culture grown from a single transformed colony 2. Incubate the cuture with vigorous shaking unti the bacteria enters the late log phase of growth (ie, an ODom of approx 0.6) 3. inoculate 500 ml of LB, YT, or Teric Broth (prewarmed to 37 C )containing the appropriate antibiotic in a 2- iter fask with ml of the late-log-phase culture Incubate the culture for approx. 2.5 ours at 3r C with vigorous shaking(250 cycles/minute on a rotary shaken) 4. For relaxed plasmids with low or moderate opy numbers, add 2.5 ml of 34 mg/ml chloramphenicol. The final concentration of chloramphenicol in the allure should be 170 glm For high-copy - iumber plasmids, do not add chloramphenicol 5. haubate the cuture 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 miarofuge tube and store it at 4'C. Harvest the remainder of the bacterial cells from the 500-ml cuture by centrifugation at 2700g(4100 pm na Sonvall GSA rotor) for 15 minutes at 4C. Discard the supematant Stand the open centrifuge bottle in an inverted postion. 1. Resuspend the bacterial pellet in 200 ml of ice-cold STE Collect the bacteria cells by centrifugation as desorbed in Step 6. Store the pellet f bacteria in the centrifuge bottle at-20C 8. Use one of the methods described in Chapter 1. Protocol 1 or Chacter 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. Alow the fozen 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 t a 30-ml plastic screw-cap tube 10. Add 2 ml of a freshly prepared lysozyme solution( 10 m /m) ollowed by 8ml of 0. 25 M EDTA (pH 8.0) IL. 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 bacteral suspension. Be as gente as possible to minimize shearing of the liberated chromosomal DNA 13. As soon as mixing is completed, add 6 ml of 5 M Nacl (fnal concentration =1 M). Use a glass rod to mix the contents of the tube gently but thoroughly. Place the tube on ice for a least 1 hour I4. Remove high-moleaular-weight DNA and bacterial debris by centifugation at 71,000g (30,000 rpm in a Beckman Type 50 rotor) for 30 minutes at 4 C. Carefuly transfer the supematant to a 50-ml disposable plas ic centifuge tube. Discard 15. Extrad the supernatant once with phenot chloroform and once with chloroform. 16. Transfer the aqueous phase to a 250-ml centrfuge bottle. Add 2 volumes (approx 60 ml) of ethanol at room temperature. Mix the solufion wel. Store the solution for 1-2 hours at room temperature. 17. Recover the nudeic acids by centrifugation at 5000g (500 rpm in a Sorval GSA rotor or 5100 pm in a Sorval HS4 swing-out rotor) for 20 minutes at4℃ 18. Discard the supematant. Wash the pellet and sides of the centrifuge tube with 70% ethanol at room temperature and then centrifuge as n Step 17 19. Discard as much of the ethanol as possible, and then inert the centrifuge bottle n a pad of paper towels to allow the last of the efhand to drain away. Use a vacum aspirator to remove droplets of ethanol fom the walls of the centrifuge otte Stand the bottle in an inverted position until no trace of ethanol is visible. At this stage, the pellet should still be 20. Dissolve the damp pellet of nudeic acid in 3 m of TE (dH 8.0) 21. Purty the crude plasmid DNA either by chromatography on commercial resins(please see Chapter 1, Protocol 9)or isopycnic centrifugation in CsCH-ethidium bromide gradients (please see Chapter 1. Protocol 10 and Chapter 1. Protocol 11). 22. Check the stuture of the plasmid by restriction enzyme digestion followed by gel electrophoresis REFERENCES htp hwwnoeaarnigcommembersproblipspprorumber 7bchenumber=1(1/2)2002-2-18 16:12111Chapter: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]