Molecular Cloning CHAPTER1>PROTOCOL12 Protocol 12 Removal of Ethidium Bromide from DNA by Extraction with 合TEH89 Nuclelc Acids METHOD Use a Past pipette to the upper (organic)phase.which is now a beaut deep pink color,to an app ion(Steps 1-4)four to six times untl all the pink color disappears fom both the aqueous phase anc CSCI-DNA S on prior to dluion with H0n5tep7力tot ec (.000 rpm ed DN then a 14 f DNA and calculate the con ation of DNA Store the DNA in aliquots at REFERENCES tienngecMheDAhetntheapetetcatns时covasny Copyright 2000.Cold Sorng Harbor Laboratory Press
Chapter:1 Protocol:12 Removal of Ethidium Bromide from DNA by Extraction with Organic Solvents CHAPTER 1 > PROTOCOL 12 printer friendly version 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. 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 Ethanol Isoamyl alcohol, saturated with H2O n-Butanol, saturated with H2O Phenol Phenol:chloroform (1:1 v/v) TE (pH 8.0) Nucleic Acids DNA sample, purified through CsCl gradient Use material from either Step 7 of Chapter 1, Protocol 10 or Step 5 of Chapter 1, Protocol 11. METHOD 1. To the solution of DNA in a glass or polypropylene tube, add an equal volume of either water-saturated n-butanol or isoamyl alcohol. Close the cap of the tube tightly. 2. Mix the organic and aqueous phases by vortexing. 3. Centrifuge the mixture at 450g (1500 rpm in a Sorvall RT-6000 centrifuge with an HL-4 rotor and 50-ml buckets) for 3 minutes at room temperature or stand the solution at room temperature until the organic and aqueous phases have separated. 4. Use a Pasteur pipette to transfer the upper (organic) phase, which is now a beautiful deep pink color, to an appropriate waste container. 5. Repeat the extraction (Steps 1-4) four to six times until all the pink color disappears from both the aqueous phase and organic phases. 6. Remove the CsCl from the DNA solution by ethanol precipitation (please follow Steps 7 through 12), by spin dialysis through a microconcentrator (Amicon), or by dialysis overnight (16 hours) against 2 liters of TE (pH 8.0) (change buffer frequently). If one of the latter two methods is used, then proceed to Step 13. 7. To precipitate the DNA from the CsCl-DNA solution, measure the volume of the CsCl solution, add three volumes of H2O, and mix the solution well. 8. Add 8 volumes of ethanol (1 volume is equal to that of the CsCl-DNA solution prior to dilution with H2O in Step 7) to the DNA solution and mix well. Store the mixture for at least 15 minutes at 4°C. 9. Collect the precipitate of DNA by centrifugation at 20,000g (13,000 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4°C. 10. Decant the supernatant to a fresh centrifuge tube. Add an equal volume of absolute ethanol to the supernatant. Store the mixture for at least 15 minutes at 4°C and then collect the precipitate of DNA by centrifugation at 20,000g (13,000 rpm in a Sorvall SS-34 rotor) for 15 minutes. 11. Wash the two DNA precipitates with 70% ethanol. Remove as much of the 70% ethanol as possible and then allow any remaining fluid to evaporate at room temperature. 12. Dissolve the precipitated DNA in 2 ml of H2O or TE (pH 8.0). 13. If the resuspended DNA contains significant quantities of ethidium bromide, as judged from its color or its emission of fluorescence when illuminated by UV light, extract the solution once with phenol and once with phenol:chloroform, and then again precipitate the DNA with ethanol. 14. Measure the OD260 of the final solution of DNA, and calculate the concentration of DNA. Store the DNA in aliquots at - 20°C. REFERENCES 1. Cozzarelli N.R., Kelly R.B., and Kornberg A. 1968. A minute circular DNA from Escherichia coli 15. Proc. Natl. Acad. Sci. 60:992-999. 2. Wang J.C. 1969. Variation of the average rotation angle of the DNA helix and the superhelical turns of covalently closed cyclic DNA. J. Mol. Biol. 43:25-39. 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=12&chpnumber=1 [2002-2-18 16:12:52]
Molecular Cloning L A BO R A T O R CHAPTER1>PROTOCOL13 ●printer friendly versio Protocol 13 Removal of Ethidium Br rom DNA by o-exchnge bromderemm DNby chromaorapyc ATERIAL 合petaewemem8e9ooange ofom (1:.w METHOD 1.Befo the Dov 00 ml of 1 N HC nue the process with with H2O (100 ml each),follo ct a 1-ml colum of Do m column volumes of TE (pH 8.0).Apply the solution c g the 2 5 col h70% ove as much as possible of the70%ethanol and then allow any TE () en tration of DNA Store the DNA in aliquots at REFERENCES x fo 2.1814:130
Chapter:1 Protocol:13 Removal of Ethidium Bromide from DNA by Ion-exchange Chromatography CHAPTER 1 > PROTOCOL 13 printer friendly version 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. 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 Ethanol HCl (1 N) NaCl (5 M) Phenol Phenol:chloroform (1:1, v/v) TE (pH 8.0) TEN buffer TEN buffer containing 0.2% sodium azide Nucleic Acids DNA sample, purified through CsCl gradient Use material from either Step 7 of Chapter 1, Protocol 10 or Step 5 of Chapter 1, Protocol 11. METHOD 1. Before using, equilibrate the Dowex AG50 resin: a. Stir approx. 20 g of Dowex AG50 in approx. 100 ml of 1 M NaCl for 5 minutes. Allow the resin to settle, and remove the supernatant by aspiration. b. Add approx. 100 ml of 1 N HCl, and stir the slurry for a further 5 minutes. Again allow the resin to settle, and remove the supernatant by aspiration. c. Continue the process with two washes with H2O (100 ml each), followed by one wash with 100 ml of TEN buffer. d. Store the equilibrated resin at 4°C in TEN buffer containing 0.2% sodium azide. 2. Construct a 1-ml column of Dowex AG50 in a Pasteur pipette. 3. Remove the buffer above the resin, and rinse the column with 2 column volumes of TE (pH 8.0). Apply the solution of DNA containing ethidium bromide and CsCl directly to the resin. 4. Immediately begin collecting the effluent from the column. After all of the DNA solution has entered the column, wash the resin with 1.2 column volumes of TE (pH 8.0) and continue to collect the eluate into a 30-ml Corex tube. 5. After the column has run dry, dilute the eluate with 2.5 column volumes of H2O. 6. Precipitate the DNA by adding eight volumes of ethanol followed by incubation for 15 minutes at 4°C. Collect the DNA by centrifugation at 17,000g (12,000 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4°C. 7. Decant the supernatant to a fresh centrifuge tube. Add an equal volume of absolute ethanol to the supernatant. Store the mixture for at least 15 minutes at 4°C and then collect the precipitate of DNA by centrifugation at 20,000g (13,000 rpm in a Sorvall SS-34 rotor) for 15 minutes. 8. Wash the two DNA precipitates with 70% ethanol. Remove as much as possible of the 70% ethanol and then allow any remaining fluid to evaporate at room temperature. 9. Dissolve the precipitated DNA in 2 ml of H2O or TE (pH 8.0). 10. If the resuspended DNA contains significant quantities of ethidium bromide, as judged from its color or its emission of fluorescence when illuminated by UV light, extract the solution once with phenol and once with phenol:chloroform, and then again precipitate the DNA with ethanol. 11. Measure the OD260 of the final solution of DNA, and calculate the concentration of DNA. Store the DNA in aliquots at - 20°C. REFERENCES 1. Radloff R., Bauer W., and Vinograd J. 1967. A dye-buoyant density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. 57:1514-1521. 2. Waring M.J. 1965. Complex formation between ethidium bromide and nucleic acids. J. Mol. Biol. 13:269-282. 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=13&chpnumber=1 [2002-2-18 16:13:00]
Molecular Cloning printe Protocol 14 MATERIALS 合 8Eane以g METHOD d DN 046:Sa1网a6occ m之
Chapter:1 Protocol:14 Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation through NaCl CHAPTER 1 > PROTOCOL 14 printer friendly version 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. 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 Ethanol NaCl (1 M) in TE (pH 8.0) Sodium acetate (3 M, pH 5.2) TE (pH 8.0) Enzymes and Buffers DNase-free Pancreatic RNase Nucleic Acids DNA sample, purified through CsCl gradient Use material from either Step 14 of Chapter 1, Protocol 12 or Step 11 of Chapter 1, Protocol 13. METHOD 1. Measure the volume of the plasmid preparation. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. Store the mixture for 30 minutes at 4°C. 2. Recover the precipitate of nucleic acids by centrifugation at >10,000g (>9100 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4°C. Decant as much of the supernatant as possible, and then store the open tube on the bench for a few minutes to allow the ethanol to evaporate. 3. Dissolve the damp pellet in 0.5-1.0 ml of TE (pH 8.0). 4. Add DNase-free RNase to a final concentration fo 10 µg/ml. Incubate the mixture for 1 hour at room temperature. 5. Add 4 ml of 1 M NaCl in TE (pH 8.0) to a Beckman SW50.1 centrifuge tube (or its equivalent). Use an automatic pipette with a disposable tip to layer up to 1 ml of the plasmid preparation on top of the 1 M NaCl solution. If necessary, fill the tube with TE (pH 8.0). 6. Centrifuge the solution at 150,000g (40,000 rpm in a Beckman SW50.1 rotor) for 6 hours at 20°C. Carefully discard the supernatant. 7. Dissolve the pellet of plasmid DNA in 0.5 ml of TE (pH 8.0). Add 50 µl of 3 M sodium acetate (pH 5.2), and transfer the DNA solution to a microfuge tube. 8. Precipitate the DNA by addition of 2 volumes of ethanol, and store the ethanolic solution for 10 minutes at 4°C. Recover the DNA by centrifugation at maximum speed for 15 minutes at 4°C in a microfuge. Decant as much of the supernatant as possible and then store the open tube on the bench for a few minutes to allow the ethanol to evaporate. 9. Dissolve the damp pellet of DNA in TE (pH 8.0). 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=14&chpnumber=1 [2002-2-18 16:13:07]
Molecular Cloning CHAPTER1>PROTOCOL 15 Protocol 15 hrwg e Acid fr s e (P TE (pH .containing RNase anp45 e the m s laye have be k8aheughaa7%agroaepelorby nutes to allo tin TE (pH 8.0) REFERENCES P
Chapter:1 Protocol:15 Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Chromatography through Sephacryl S-1000 CHAPTER 1 > PROTOCOL 15 printer friendly version 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. 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 Bromophenol blue sucrose solution Ethanol Phenol Sephacryl equilibration buffer Sodium acetate (3 M, pH 5.2) TE (pH 8.0) containing 20 µg/ml RNase A Nucleic Acids DNA sample, purified through CsCl gradient Use material from either Step 14 of Chapter 1, Protocol 12 or Step 11 of Chapter 1, Protocol 13 . METHOD 1. Prepare a 1 x 10-cm column of Sephacryl S-1000, equilibrated in Sephacryl equilibration buffer. 2. Measure the volume of the plasmid preparation. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. Store the mixture for 30 minutes at 4°C. 3. Recover the precipitate of nucleic acids by centrifugation at >10,000g (>9100 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4°C. Drain off as much of the supernatant as possible, and then store the open tube on the bench for a few minutes to allow the ethanol to evaporate. 4. Dissolve the damp pellet of nucleic acids in a small volume (<400 µl) of TE (pH 8.0) containing RNase A at a final DNA concentration of at least 100 µg/ml. 5. Incubate the mixture for 1 hour at room temperature. 6. Extract the solution once with an equal volume of phenol equilibrated in TE (pH 8.0). 7. Recover the aqueous layer, and add 100 µl of bromophenol blue dye sucrose solution. Layer the blue aqueous phase on the column of Sephacryl S-1000. 8. Wash the DNA into the column, and apply a reservoir of Sephacryl equilibration buffer. Immediately begin collecting 0.5- ml fractions. 9. When 15 fractions have been collected, clamp off the bottom of the column. At this stage, the blue dye should have traveled about half the length of the column. 10. Analyze 10 µl of each fraction by electrophoresis through a 0.7% agarose gel or by ethidium bromide fluorescence to identify the fractions containing plasmid DNA. 11. Pool the fractions containing plasmid DNA, and recover the DNA by precipitation with 2 volumes of ethanol for 10 minutes at 4°C and centrifugation at 10,000g (9200 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4°C. 12. Decant as much of the supernatant as possible, and then store the open tube on the bench for a few minutes to allow the ethanol to evaporate. 13. Dissolve the damp pellet in TE (pH 8.0). REFERENCES 1. Gomez-Marquez J., Freire M., and Segade F. 1987. A simple procedure for the large-scale purification of plasmid DNA. Gene 54:255-259. 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=15&chpnumber=1 [2002-2-18 16:13:19]
Molecular Cloning ●printer friendy ve Protocol 16 Ramomoisme ay黑on ons of plasm NA by high MATERIALS te (3 M.pH 5.2) METHOD (pH 5)and 2 volmes of e230022o92as840em5。 tube and add 6 m of isopropanol.Allow the plasmid DNA to precip the tube open tube on the bench top for a fow minutes unti the ethanol has amp pollet of DNA in TE (pH 8.0) REFERENCES AP. R.ar
Chapter:1 Protocol:16 Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Lithium Chloride CHAPTER 1 > PROTOCOL 16 printer friendly version 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. 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 Ethanol Isopropanol LiCl (4 M) Sodium acetate (3 M, pH 5.2) TE (pH 8.0) TE (pH 8.0) containing 20 µg/ml RNase A Nucleic Acids DNA sample, purified through CsCl gradient Use material from either Step 14 of Chapter 1, Protocol 12 or Step 11 of Chapter 1, Protocol 13. METHOD 1. Measure the volume of the plasmid preparation. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. Store the mixture for 30 minutes at 4°C. 2. Recover the precipitate of nucleic acids by centrifugation at >10,000g (>9100 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4°C. Drain off as much of the supernatant as possible, and then store the open tube on the bench for a few minutes to allow the ethanol to evaporate. 3. Dissolve the damp pellet in 1 ml of TE (pH 8.0) containing RNase A at a concentration of 100 µg/ml. 4. Add 3 ml of 4 M LiCl solution. Incubate the mixture on ice for 30 minutes. 5. Separate the plasmid DNA from the precipitated nucleic acids by centrifugation at 12,000g (10,000 rpm in a Sorvall SS- 34 rotor) for 15 minutes at 4°C. 6. Transfer the supernatant to a fresh centrifuge tube and add 6 ml of isopropanol. Allow the plasmid DNA to precipitate for 30 minutes at room temperature. 7. Recover the precipitated plasmid DNA by centrifugation at 12,000g (10,000 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4°C. 8. Carefully remove the supernatant and add 5-10 ml of 70% ethanol to the tube. Vortex the tube briefly, and then recentrifuge at 12,000g for 10 minutes at 4°C. 9. Carefully remove the supernatant, and store the open tube on the bench top for a few minutes until the ethanol has evaporated. 10. Dissolve the damp pellet of DNA in TE (pH 8.0). REFERENCES 1. Barlow J.J., Mathias A.P., Williamson R., and Gammack D.B. 1963. A simple method for quantitative isolation of undegraded high molecular weight ribonucleic acid. Biochem. Biophys. Res. Commun. 13:61-66. 2. Kondo T., Mukai M., and Kondo Y. 1991. Rapid isolation of plasmid DNA and LiCl-ethidium bromide treatment and gel filtration. Anal. Biochem. 198:30-35. 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=16&chpnumber=1 [2002-2-18 16:13:33]