@片Molecular Cloning L A BO R A T O R CHAPTER1>PROTOCOL17 Protocol 17 Directional Cloning into Plas 6ws ocol 23 col 24,Chap METHOD el in TE A and D 1.0 MM ATP ON ATP 10 af with d Tube DNA 2105
Chapter:1 Protocol:17 Directional Cloning into Plasmid Vectors CHAPTER 1 > PROTOCOL 17 printer friendly version Protocol 17 Directional Cloning into Plasmid Vectors Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes that generate incompatible termini and that the fragment of DNA to be cloned carries termini that are compatible with those of the doubly cleaved vector. 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 ATP (10 mM) Omit ATP from the ligation reaction in Step 5 if the ligation buffer contains ATP. Ethanol Phenol:chloroform (1:1, v/v) Sodium acetate (3 M, pH 5.2) TE (pH 8.0) Enzymes and Buffers Bacteriophage T4 DNA ligase Restriction endonucleases Nucleic Acids Vector DNA (plasmid) Target DNA fragment Adaptors may be added to the target DNA as described in Chapter 1, Protocol 18 . Additional Reagents Step 7 of this protocol requires the reagents listed in Chapter 1, Protocol 23 , Chapter 1, Protocol 24 , Chapter 1, Protocol 25 , or Chapter 1, Protocol 26 . METHOD 1. Digest the vector (10 µg) and foreign DNA with the two appropriate restriction enzymes. 2. Purify the digested foreign DNA by extraction with phenol:chloroform and standard ethanol precipitation. 3. Purify the vector DNA by spun-column chromatography followed by standard ethanol precipitation. 4. Reconstitute the precipitated DNAs separately in TE (pH 8.0) at a concentration of approx. 100 µg/ml. Calculate the concentration of the DNA (in pmole/ml), assuming that 1 bp has a mass of 660 daltons. 5. Transfer appropriate amounts of the DNAs to sterile 0.5-ml microfuge tubes as follows: Tube DNA A and D vector (30 fmoles [approx. 100 ng]) B insert (foreign) (30 fmoles [approx. 10 ng]) C and E vector (30 fmoles) plus insert (foreign) (30 fmoles) F superhelical vector (3 fmoles [approx. 10 ng]) The molar ratio of plasmid vector to insert DNA fragment should be approx. 1:1 in the ligation reaction. The final DNA concentration should be approx. 10 ng/µl. a. To Tubes A, B, and C add: 10x Ligation buffer 1.0 µl Bacteriophage T4 DNA ligase 0.1 Weiss unit 10 mM ATP 1.0 µl H2O to 10 µl b. To Tubes D and E, add: 10x Ligation buffer 1.0 µl 10 mM ATP 1.0 µl H2O to 10 µl no DNA ligase The DNA fragments can be added to the tubes together with the H2O and then warmed to 45°C for 5 minutes to melt any cohesive termini that have reannealed during fragment preparation. Chill the DNA solution to 0°C before the remainder of the ligation reagents are added. 6. Incubate the reaction mixtures overnight at 16°C or for 4 hours at 20°C. 7. Transform competent E. coli with dilutions of each of the ligation reactions as described in Chapter 1, Protocol 23 , Chapter 1, Protocol 24 , Chapter 1, Protocol 25 , or Chapter 1, Protocol 26 . As controls, include known amounts of a standard preparation of superhelical plasmid DNA to check the efficiency of transformation. Tube DNA Ligase Expected number of transformed colonies A vector + approx. 0 (approx. 104 fewer than Tube F) B insert + 0 C vector and insert + approx. 10-fold more then Tube A or D D vector - approx. 0 (approx. 104 fewer than Tube F) E vector and insert - some, but fewer than Tube C F superhelical vector - >2 x 105 printer friendly version http://www.molecularcloning.com/members/protocol.jsp?pronumber=17&chpnumber=1 (1 / 2) [2002-2-18 16:13:42]
Molecular Cloning CHAPTER1>PROTOCOL1 Protocol 18 MATERIALS chion in Step 2 the butter contains ATP METHOD 2rnEn MATP 1.0 0, 10 me as possible (5-10 l). r.Add sterile H,O to a final volum n r done ary to ●rintev aaaIOaeI
Chapter:1 Protocol:18 Attaching Adaptors to Protruding Termini CHAPTER 1 > PROTOCOL 18 printer friendly version Protocol 18 Attaching Adaptors to Protruding Termini Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms. 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 ATP (10 mM) Omit ATP from the ligation reaction in Step 2 if the ligation buffer contains ATP. Ethanol Phenol:chloroform (1:1, v/v) Sodium acetate (3 M, pH 5.2) TE (pH 8.0) Enzymes and Buffers Bacteriophage T4 DNA ligase 10x Linker kinase buffer Polynucleotide kinase Restriction endonucleases Nucleic Acids and Oligonucleotides Target DNA fragment Synthetic oligonucleotide/adaptor dissolved in TE-(pH 8.0) at a concentration of approx. 400 µg/ml. For a hexamer, this concentration is equivalent to a 50 µM solution METHOD 1. To phosphorylate the adaptors, add to a sterile microfuge tube: synthetic oligonucleotide or adaptor 0.5-2.0 µg, dissolved in TE (pH 8.0) 10x linker kinase buffer 1.0 µl 10 mM ATP 1.0 µl H2O to 10 µl Bacteriophage T4 polynucleotide kinase 1.0 unit Incubate the reaction for 1 hour at 37°C. 2. To ligate the phophorylated adaptors to a DNA fragment with complementary protruding ends, set up a ligation reaction as follows: DNA fragment 100-200 ng phosphorylated adapter 10-20-fold molar excess 10x ligation buffer 1.0 µl bacteriophage T4 DNA ligase 0.1 Weiss unit 10 mM ATP 1.0 µl H2O to 10 µl Incubate the ligation mixture for 6-16 hours at 4°C. To achieve the maximum efficiency of ligation, set up the reactions in as small a volume as possible (5-10 µl). 3. Inactivate the DNA ligase by incubating the reaction mixture for 15 minutes at 65°C. 4. Dilute the ligation reaction with 10 µl of the appropriate 10x restriction enzyme buffer. Add sterile H2O to a final volume of 100 µl followed by 50-100 units of restriction enzyme. 5. Incubate the reaction for 1-3 hours at 37°C. 6. Extract the restriction digestion with phenol:chloroform and recover the DNA by standard ethanol precipitation. 7. Collect the precipitated DNA by centrifugation at maximum speed for 15 minutes at 4°C in a microfuge, and resuspend the DNA in 50 µl of TE (pH 8.0). 8. Pass the resuspended DNA through a spun column to remove excess adaptors and their cleavage products. 9. The modified DNA fragment can now be ligated to a plasmid vector with protruding ends that are complementary to those of the cleaved adaptor (please see Chapter 1, Protocol 17 ). 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=18&chpnumber=1 [2002-2-18 16:13:50]
Molecular Cloning L A BO R A T O R CHAPTER1>PROTOCOL19 printer friendy versio Protocol 19 Blunt-nded Cloning into Plasmid Vector and the are usedt the ligation reaction in Step 5 the figation buffer contains ATP m(1:1.w) TE (PH8.0 lucloic Acid METHOD 1-10 ug of thesm DNA and foreign DNA with the appropriat mestrk n DNA and vector DNA by extraction with phenobchioroform and standard ethano Reg 1 Aand E 1(60 mmoles lappeox.100 ngD Cand F ign (60 a.To TubesA B.and Cadd: age T4 DNA Tgase iss un? 0 30%PE8000 To Tubes D.E and at 16'C or for 4 hours al 20C DNA
Chapter:1 Protocol:19 Blunt-ended Cloning into Plasmid Vectors CHAPTER 1 > PROTOCOL 19 printer friendly version Protocol 19 Blunt-ended Cloning into Plasmid Vectors Target DNA is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent E. coli. The maximum number of "correct" clones can generally be obtained from ligation reactions containing equimolar amounts of plasmid and target DNAs, with the total DNA concentration being <100 µg/ml. Bluntend ligation catalyzed by bacteriophage T4 DNA ligase is suppressed by high concentrations (5 mM) of ATP and polyamines such as spermidine. 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 ATP (10 mM) Omit ATP from the ligation reaction in Step 5 if the ligation buffer contains ATP. Ethanol Phenol:chloroform (1:1, v/v) PEG 8000 (30% w/v) Sodium acetate (3 M, pH 5.2) TE (pH 8.0) Enzymes and Buffers Bacteriophage T4 DNA ligase Restriction endonucleases Nucleic Acids Target DNA (blunt-end fragment) Vector DNA (plasmid) Additional Reagents Step 4 of this protocol requires the reagents listed in Chapter 1, Protocol 20. Step 7 of this protocol requires the reagents listed in Chapter 1, Protocol 23 , Chapter 1, Protocol 24 , Chapter 1, Protocol 25 , or Chapter 1, Protocol 26 . METHOD 1. In separate reactions, digest 1-10 µg of the plasmid DNA and foreign DNA with the appropriate restriction enzyme(s) that generate blunt ends. 2. Purify the digested foreign DNA and vector DNA by extraction with phenol:chloroform and standard ethanol precipitation. 3. Reconstitute the precipitated DNAs separately in TE (pH 8.0) at a concentration of approx. 100 µg/ml. Calculate the concentration of the DNAs (in pmole/ml) assuming that 1 bp has a mass of 660 daltons. 4. Dephosphorylate the plasmid vector DNA as described in Chapter 1, Protocol 20 . 5. Transfer appropriate amounts of the DNAs to sterile 0.5-ml microfuge tubes as follows: Tube DNA A and E vector1 (60 fmoles [approx. 100 ng]) B foreign2 (60 fmoles [approx. 10 ng]) C and F vector1 (60 fmoles) plus foreign (60 fmoles)3 D linearized vector (contains 5'-terminal phosphates) (60 fmoles) G superhelical vector (6 fmoles [approx. 10 ng]) 1Vector DNA is dephosphorylated as described in Chapter 1, Protocol 20 . 2Linkers may be ligated to foreign target DNA. 3The molar ratio of plasmid vector to insert DNA fragment should be approx. 1:1 in the ligation reaction. The total DNA concentration in the ligation reaction should be approx. 10 ng/µl. a. To Tubes A, B, and C add: 10x Ligation buffer 1.0 µl Bacteriophage T4 DNA ligase 0.5 Weiss unit 5 mM ATP 1.0 µl H2O to 8.5 µl 30% PEG 8000 1-1.5 µl b. To Tubes D, E, and F add: 10x Ligation buffer 1.0 µl 5 mM ATP 1.0 µl H2O to 8.5 µl 30% PEG 8000 1-1.5 µl no DNA ligase To achieve the maximum efficiency of ligation, set up the reactions in as small a volume as possible (5-10 µl). The DNA fragments can be added to the tubes together with the H2O and then warmed to 45°C for 5 minutes to help dissociate any clumps of DNA that have formed during fragment preparation. Chill the DNA solution to 0°C before the remainder of the ligation reagents are added. 6. Incubate the reaction mixtures overnight at 16°C or for 4 hours at 20°C. 7. Transform competent E. coli with dilutions of each of the ligation reactions, using one of the methods described in Chapter 1, Protocol 23, Chapter 1, Protocol 24, Chapter 1, Protocol 25 , or Chapter 1, Protocol 26 . As controls, include known amounts of a standard preparation of superhelical plasmid DNA to check the efficiency of transformation. Tube DNA Ligase Expected number of transformants http://www.molecularcloning.com/members/protocol.jsp?pronumber=19&chpnumber=1 (1 / 2) [2002-2-18 16:13:59]
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Chapter:1 Protocol:19 Blunt-ended Cloning into Plasmid Vectors A vector1 + approx. 03 B insert + 0 C vector1 and insert + approx. 5-fold more then Tube F D vector1 - approx. 0 E vector2 - approx. 50-fold more than Tube D F vector1 and insert - approx. 50-fold more than Tube D G superhelical vector - 2 x 105 1Dephosphorylated 2Not dephosphorylated 3Transformants arising from ligation of dephosphorylated vector DNA alone are due to failure to remove 5' residues during treatment with alkaline phosphatase. REFERENCES 1. Bercovich J.A., Grinstein S., and Zorzopulos J. 1992. Effect of DNA concentration of recombinant plasmid recovery after blunt-end ligation. BioTechniques 12:190-193. 2. Ferretti L. and Sgaramella V. 1981. Temperature dependence of the joining by T4 DNA ligase of termini produced by type II restriction endonucleases. Nucleic Acids Res. 9:85-93. 3. Sgaramella V. and Ehrlich S.D. 1978. Use of the T4 polynucleotide ligase in the joining of flush-ended DNA segments generated by restriction endonucleases. Eur. J. Biochem. 86:531-537. 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=19&chpnumber=1 (2 / 2) [2002-2-18 16:13:59]
Molecular Cloning L A BO R A T OR Y Protocol 20 Dephosphorylation of Plasmid DNA T4 DN .. n(1:1.wo △⊙De%aMpH52sdpH7a 8 METHOD ss of the desiro m and recover the DNA by standard s. per mole DNA Ends Te Time 55C/45 minu Blunt 0.2 SAP Ea9nE87酒omr807oo65Gc0mnesr75cr0mno时nepes6neeo5 on t10re he ONAt0C REFERENCES 1977. Ttm1t/n3e034w1wt网
Chapter:1 Protocol:20 Dephosphorylation of Plasmid DNA CHAPTER 1 > PROTOCOL 20 printer friendly version Protocol 20 Dephosphorylation of Plasmid DNA During ligation in vitro, T4 DNA ligase will catalyze the formation of a phosphodiester bond between adjacent nucleotides only if one nucleotide carries a 5´-phosphate residue and the other carries a 3´-hydroxyl terminus. Recircularization of vector DNA can therefore be minimized by removing the 5´-phosphate residues from both termini of the linear, double-stranded plasmid DNA with alkaline phosphatase. 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 EDTA (0.5 M, pH 8.0) EGTA (0.5 M, pH 8.0) Ethanol Phenol Phenol:chloroform (1:1, v/v) SDS (10% w/v) Sodium acetate (3 M, pH 5.2 and pH 7.0) TE (pH 8.0) Tris-Cl (10 mM, pH 8.3) Enzymes and Buffers Calf intestinal alkaline phosphatase (CIP) Shrimp alkaline phosphatase (SAP) Proteinase K (10 mg/ml) Restriction endonucleases Nucleic Acids Vector DNA (closed circular plasmid) METHOD 1. Digest a reasonable quantity of closed circular plasmid DNA (10 µg) with a two- to threefold excess of the desired restriction enzyme for 1 hour. 2. Remove an aliquot (0.1 µg), and analyze the extent of digestion by electrophoresis through a 0.7% agarose gel containing ethidium bromide, using undigested plasmid DNA as a marker. If digestion is not complete, add more restriction enzyme and continue the incubation. 3. When digestion is complete, extract the sample once with phenol:chloroform and recover the DNA by standard precipitation with ethanol. Store the ethanolic solution on ice for 15 minutes. 4. Recover the DNA by centrifugation at maximum speed for 10 minutes at 4°C in a microfuge, and dissolve the DNA in 110 µl of 10 mM Tris-Cl (pH 8.3). 5. To the remaining 90 µl of the linearized plasmid DNA, add 10 µl of 10x CIP or 10x SAP buffer and the appropriate amount of calf intestinal phosphatase (CIP) or shrimp alkaline phosphatase (SAP) and incubate as described in the table below. Conditions for Dephosphorylation of 5´-phosphate Residues from DNA Type of Enzyme/Amount Incubation Terminus per mole DNA Ends Temperature/Time 5´-Protruding 0.01 unit CIPa 37°C/30 minutes 0.1 unit SAP 37°C/60 minutes 3´-Protruding 0.1-0.5 unit CIPb 37°C/15 minutes then 55°C/45 minutes 0.5 unit SAP 37°C/60 minutes Blunt 0.1-0.5 unit CIPb 37°C/15 minutes then 55°C/45 minutes 0.2 unit SAP 37°C/60 minutes aAfter the initial 30-minute incubation, add a second aliquot of CIP enzyme and continue incubation for another 30 minutes at 37°C. bAdd a second aliquot of CIP just before beginning the incubation at 55°C. 6. Inactivate the phosphatase activity: To inactivate CIP at the end of the incubation period: Add SDS and EDTA (pH 8.0) to final concentrations of 0.5% and 5 mM, respectively. Mix well, and add proteinase K to a final concentration of 100 µg/ml. Incubate for 30 minutes at 55°C. Alternatively, CIP can be inactivated by heating to 65°C for 30 minutes (or 75°C for 10 minutes) in the presence of 5 mM EDTA or 10 mM EGTA (both at pH 8.0). or To inactivate SAP: Incubate the reaction mixture for 15 minutes at 65°C in the dephosphorylation buffer. 7. Cool the reaction mixture to room temperature, and then extract it once with phenol and once with phenol:chloroform. 8. Recover the DNA by standard precipitation with ethanol. Mix the solution again and store it for 15 minutes at 0°C. 9. Recover the DNA by centrifugation at maximum speed for 10 minutes at 4°C in a microfuge. Wash the pellet with 70% ethanol at 4°C and centrifuge again. 10. Carefully remove the supernatant and leave the open tube on the bench to allow the ethanol to evaporate. 11. Dissolve the precipitated DNA in TE (pH 8.0) at a concentration of 100 µg/ml. Store the DNA in aliquots at -20°C. REFERENCES 1. Seeburg P., Shine J., Martial J.A., Baxter J.D., and Goodman H.M. 1977. Nucleotide sequence and amplification in http://www.molecularcloning.com/members/protocol.jsp?pronumber=20&chpnumber=1 (1 / 2) [2002-2-18 16:14:29]