a,8 ae1Oo Copyright2000.Cold Spring Harbor Laboratsry Press
Chapter:1 Protocol:7 Preparation of Plasmid DNA by Lysis with SDS 1. Godson G.N. and Vapnek D. 1973. A simple method for the large-scale purification of øX174 RFI supercoiled DNA. Biochim. Biophys. Acta 299:516-520. 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=7&chpnumber=1 (2 / 2) [2002-2-18 16:12:11]
Molecular Cloning CHAPTER1>PROTOCOL。 Protocol8 MATERIALS 0 TE(H)containing 20 RNaseA METHOD 1.are-scaemd c nencetcherotmanoewhchlook NA 310 d for 5 mi 020 13. REFERENCES of DNA-fraoments by polvethylene glycol in nized PEG-methed for rapid DNA:Hich vicid fron ee2e Copyright2000.Cold Spring Hartor Laboratery Press
Chapter:1 Protocol:8 Purification of Plasmid DNA by Precipitation with Polyethylene Glycol CHAPTER 1 > PROTOCOL 8 printer friendly version Protocol 8 Purification of Plasmid DNA by Precipitation with Polyethylene Glycol Crude preparations of plasmid DNA are first treated with lithium chloride and RNase (to remove RNA). The plasmid DNA is then precipitated in a solution containing polyethylene glycol and MgCl2. 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 Chloroform Ethanol Isopropanol LiCl (5 M) PEG-MgCl2 solution Phenol:chloroform (1:1, v/v) Sodium acetate (3 M, pH 5.2) TE (pH 8.0) TE (pH 8.0) containing 20 µg/ml RNase A Nucleic Acids Crude plasmid preparation Use material from either Step 17 of Chapter 1, Protocol 3 or Step 19 of Chapter 1, Protocol 5 . METHOD 1. Transfer 3 ml of the crude large-scale plasmid preparation to a 15-ml Corex tube and chill the solution to 0°C in an ice bath. 2. Add 3 ml of an ice-cold solution of 5 M LiCl to the crude plasmid preparation, mix well, and centrifuge the solution at 12,000g (10,000 rpm in a Sorvall SS-34 rotor) for 10 minutes at 4°C. 3. Transfer the supernatant to a fresh 30-ml Corex tube. Add an equal volume of isopropanol. Mix well. Recover the precipitated nucleic acids by centrifugation at 12,000g (10,000 rpm in a Sorvall SS-34 rotor) for 10 minutes at room temperature. 4. Decant the supernatant carefully, and invert the open tube to allow the last drops of supernatant to drain away. Rinse the pellet and the walls of the tube with 70% ethanol at room temperature. Carefully discard the bulk of the ethanol, and then use a vacuum aspirator to remove any beads of liquid that adhere to the walls of the tube. Place the inverted, open tube on a pad of paper towels for a few minutes. The pellet should still be damp. 5. Dissolve the damp pellet of nucleic acid in 500 µl of TE (pH 8.0) containing RNase A. Transfer the solution to a microfuge tube and store it for 30 minutes at room temperature. 6. Extract the plasmid-RNase mixture once with phenol:chloroform and once with chloroform. 7. Recover the DNA by standard ethanol precipitation. 8. Dissolve the pellet of plasmid DNA in 1 ml of sterile H2O, and then add 0.5 ml of PEG-MgCl2 solution. 9. Store the solution for 10 minutes at room temperature, and then collect the precipitated plasmid DNA by centrifugation at maximum speed for 20 minutes at room temperature in a microfuge. 10. Remove traces of PEG by resuspending the pellet of nucleic acid in 0.5 ml of 70% ethanol. Collect the nucleic acid by centrifugation at maximum speed for 5 minutes in a microfuge. 11. Remove the ethanol by aspiration and repeat Step 10. Following the second rinse, store the open tube on the bench for 10-20 minutes to allow the ethanol to evaporate. 12. Dissolve the damp pellet in 500 µl of TE (pH 8.0). Measure the OD260 of a 1:100 dilution in TE (pH 8.0) of the solution, and calculate the concentration of the plasmid DNA assuming that 1 OD260 = 50 µg of plasmid DNA/ml. 13. Store the DNA in aliquots at -20°C. REFERENCES 1. Lis J.T. 1980. Fractionation of DNA-fragments by polyethylene glycol induced precipitation. Methods Enzymol. 65:347- 353. 2. Nicoletti V.G. and Condorelli D.F. 1993. Optimized PEG-method for rapid plasmid DNA purification: High yield from "midiprep." BioTechniques 14:532-534, 536. 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=8&chpnumber=1 [2002-2-18 16:12:19]
@片Molecular Cloning CHAPTER1>PROTOCOL printer friendy vers Protocol 9
Chapter:1 Protocol:9 Purification of Plasmid DNA by Chromatography CHAPTER 1 > PROTOCOL 9 printer friendly version Protocol 9 Purification of Plasmid DNA by Chromatography The following table summarizes the salient features of many of the commercial resins that are currently available for plasmid purification. Individual manufacturers supply detailed instructions, which should be followed to the letter. METHOD 1. Commercially Available Resins and Their Uses Resin Manufacturer Chemistry Use Notes Qiagen Qiagen macroporous silica transfection of eukaryotic some batch-to-batch variation; www.qiagen.com gel, anion-exchange cells pH-sensitive (DEAE) QIAprep Qiagen silica gel enzymic manipulation different columns available for www.qiagen.com purification of doubleor singlestranded DNAs Wizard Promega silica particle additional ethanol inexpensive, reproducible www.promega.com precipitation required for transfection of eukaryotic cells FlexiPrep Pharmacia anion exchange enzymic manipulation transfection requires further www.apbiotech.com purification Glass-Max Life Technologies silica matrix enzymic manipulation minipreps only www.lifetech.com GeniePrep Ambion hydrophobic inter- enzymic manipulation miniprep only; made in Texas www.ambion.com actions, glass fiber Perfect Prep Eppendorf 5 Prime silica matrix transfection of eukaryotic very fast; miniprep only www.5prime.com cells ClearCut Stratagene silica resin, hydro- enzymic manipulation can be used for miniprep plasMiniprep Kit www.stratagene.com phobic interaction mid or DNA fragment purification Concert, rapid Life Technologies silica gel enzymic manipulation mini- and maxipreps and high purity www.lifetech.com and transfection of systems eukaryotic cells NucleoBond AX Nest Group, Inc. macroporous silica transfection of eukaryotic five column sizes; resin good for world.std/approx. nestgrp/ gel anion exchange cells large DNA purification, including cosmids and P1 DNAs 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=9&chpnumber=1 [2002-2-18 16:12:26]
@≈一Molecular Cloning Protocol 10 二 ATERIAL 合enar Adaonepeginpoeolreqr stednChaplerl,Potbcali2crCeptar1,Proiecol13 METHOD pe so 01219 then attach a piece of Scold RENCES ive dyes.I.The 218121 2000.Cold Sering Harb
Chapter:1 Protocol:10 Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous Gradients CHAPTER 1 > PROTOCOL 10 printer friendly version Protocol 10 Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous Gradients Solutions containing plasmid DNA are adjusted to a density of 1.55 g/ml with solid CsCl. The intercalating dye, ethidium bromide, which binds differentially to closed circular and linear DNAs, is then added to a concentration of 200 µg/ml. During centrifugation to equilibrium, the closed circular DNA and linear DNAs form bands at different densities. 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 CsCl (solid) Ethanol Ethidium bromide (10 mg/ml) Paraffin oil Nucleic Acids Crude plasmid preparation Additional Reagents Step 8 of this protocol requires the reagents listed in Chapter 1, Protocol 12 or Chapter 1, Protocol 13 . METHOD 1. Measure the mass of the crude plasmid DNA preparation. Measurement is best done by transferring the solution into a fresh tube that has been tared on a top-loading balance. For every gram of plasmid DNA solution, add exactly 1.01 g of solid CsCl. Close the top of the tube to prevent evaporation and then warm the solution to 30°C to facilitate the dissolution of the CsCl salt. Mix the solution gently until the salt is dissolved. 2. Add 100 µl of 10 mg/ml ethidium bromide for each 5 g of original DNA solution. 3. If Corex glass tubes are used, centrifuge the solution at 7700g (8000 rpm in a Sorvall SS-34 rotor) for 5 minutes at room temperature. If disposable polypropylene tubes are used, centrifuge at 1100g (3000 rpm in a Sorvall SS-34 rotor) for 10 minutes. 4. Use a Pasteur pipette or a disposable syringe fitted with a large-gauge needle to transfer the clear, red solution under the scum and above the pellet to a tube suitable for centrifugation in an ultracentrifuge rotor. Top off the partially filled centrifuge tubes with light paraffin oil or rebanding solution. Make sure that the weights of tubes opposite each other in the rotor are equal. Seal the tubes according to the manufacturer's instructions. 5. Centrifuge the density gradients at 20°C as appropriate for the rotor: Beckman NVT 65 rotor 366,000g (62,000 rpm) for 6 hours Beckman VTi65 rotor 194,000g (45,000 rpm) for 16 hours Beckman Type 50Ti rotor 180,000g (45,000 rpm) for 48 hours Beckman Type 65Ti rotor 314,000g (60,000 rpm) for 24 hours Beckman Type 70.1Ti rotor 331,000g (60,000 rpm) for 24 hours 6. Gently remove the rotor from the centrifuge and place it on a flat surface. Carefully remove each tube and place it in a test tube rack covered with tin foil. In a dimly lit room (i.e., with the overhead fluorescent lights turned off), mount one tube in a clamp attached to a ring stand. 7. Collect the band of closed circular DNA. a. Use a 21-gauge hypodermic needle to make a small hole in the top of the tube to allow air to enter when fluid is withdrawn. b. Carefully wipe the outside of the tube with ethanol to remove any grease or oil, and then attach a piece of Scotch Tape or Time tape to the outside of the tube. c. Attach a 5-10-cc disposable syringe to a sterile 18-gauge hypodermic needle and insert the needle (beveled side up) into the tube through the tape so that the open, beveled side of the needle is positioned just below the lower DNA band (closed circular plasmid DNA). d. Slowly withdraw the plasmid DNA, taking care not to disturb the upper viscous band of chromosomal DNA. 8. Remove ethidium bromide from the DNA as described in one of the methods presented in Chapter 1, Protocol 12 or Chapter 1, Protocol 13 . REFERENCES 1. Bauer W. and Vinograd J. 1968. The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye. J. Mol. Biol. 33:141-171. 2. Bauer W. and Vinograd J. 1971. The use of intercalative dyes in the study of closed circular DNA. Progr. Mol. Subcell. Biol. 2:181-215. 3. 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. 4. Vinograd J. and Leibowitz J. 1966. Physical and topological properties of circular DNA. J. Gen. Physiol. 49:103-125. 5. Waring M.J. 1966. Structural requirements for the binding between ethidium bromide and nucleic acids. Biochim. Biophys. Acta 114:234-244. 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=10&chpnumber=1 [2002-2-18 16:12:34]
Molecular Cloning printerv Protocol 11 MATERIALS (m TE (pH 8.0) Nucleic Acids METHOD C488 1.Prepa 10 20 g of CsCI in 8 ml of TE 1.4052 4g of CsCl in 7 y 10 tube by layering th make a small hole in the top of the tube to allow air to enter when fuid is of the tut REFERENCES s in the pre printer friendly versio e
Chapter:1 Protocol:11 Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous Gradients CHAPTER 1 > PROTOCOL 11 printer friendly version Protocol 11 Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous Gradients A solution containing plasmid DNA, saturating amounts of ethidium bromide, and CsCl (44% w/v) is layered between two solutions of lesser (35% w/v CsCl) and greater density (59% w/v CsCl). During centrifugation to equilibrium, the closed circular plasmid DNA and linear DNAs form bands at different densities. 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 CsCl (solid) Ethidium bromide (10 mg/ml) TE (pH 8.0) Nucleic Acids Crude plasmid preparation Use material from either Step 16 of Chapter 1, Protocol 3 , Step 18 of Chapter 1, Protocol 5 , or Step 19 of Chapter 1, Protocol 7. Additional Reagents Step 6 of this protocol requires the reagents listed in Chapter 1, Protocol 12 or Chapter 1, Protocol 13 . METHOD 1. Prepare CsCl layers for a three-step discontinuous gradient as described in the table. Use a 3-cc hypodermic syringe equipped with an 18-gauge bone marrow (10 cm) needle to transfer 1.5 ml of the top layer (35%) CsCl solution to a 5- ml polyallomer ultracentrifuge tube (Beckman Quick-Seal or equivalent). Three-step Discontinuous Gradient Layers Molarity CsCl Refractive Layer (w/w) Index Preparation Top layer 2.806 1.3670 Dissolve 4.720 g of CsCl in 8 ml of TE (35%) (pH 8.0). Adjust the volume to exactly 10 ml. Then add 120 µl of 10 mg/ml ethidium bromide. Middle layer 3.870 1.3792 Dissolve 0.8 g of CsCl in exactly 1 ml of (44%) the crude DNA preparation. Then add 30 µl of 10 mg/ml ethidium bromide. Bottom layer 6.180 1.4052 Dissolve 10.4 g of CsCl in 7 ml of TE. (59%) Adjust the volume to exactly 10 ml. Then add 120 µl of 10 mg/ml ethidium bromide. 2. Use a 1-cc tuberculin syringe equipped with an 18-gauge bone marrow (10 cm) needle to layer 0.5 ml of the middle layer (44%) CsCl solution, containing the plasmid DNA, into the bottom of the tube under the top layer solution. As a rule of thumb, the crude plasmid DNA prepared from no more than 50 ml of an overnight culture should be used per gradient. The crude plasmid preparation from a 100-ml culture should be reconstituted in approx. 0.9 ml of TE (pH 8.0), which is enough to form the middle layer of two discontinuous gradients. 3. Use a 5-cc hypodermic syringe equipped with an 18-gauge bone marrow (10 cm) needle to fill the tube by layering the bottom layer (59%) CsCl solution under the middle layer CsCl solution. 4. Centrifuge the sealed tubes at 330,000g (60,000 rpm in a Beckman Type 70.1Ti rotor) for 5 hours. Make sure that the weights of tubes opposite each other in the rotor are equal. Seal the tubes according to the manufacturer's instructions. 5. Collect the band of closed circular DNA: a. Use a 21-gauge hypodermic needle to make a small hole in the top of the tube to allow air to enter when fluid is withdrawn. b. Carefully wipe the outside of the tube with ethanol to remove any grease or oil, and then attach a piece of Scotch Tape or Time tape to the outside of the tube. c. Attach a 5-10-cc disposable syringe to a sterile 18-gauge hypodermic needle and insert the needle (beveled side up) into the tube through the tape so that the open, beveled side of the needle is positioned just below the lower DNA band (closed circular plasmid DNA). d. Slowly withdraw the plasmid DNA, taking care not to disturb the upper viscous band of chromosomal DNA. 6. Remove ethidium bromide from the DNA as described in one of the methods presented in Chapter 1, Protocol 12 or Chapter 1, Protocol 13 . REFERENCES 1. Dorin M. and Bornecque C.A. 1995. Fast separations of plasmid DNA using discontinuous gradients in the preparative ultracentrifuge. BioTechniques 18:90-91. 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=11&chpnumber=1 [2002-2-18 16:12:44]