Manual: QuikChange® Site-Directed Mutagenesis Kit
Contents
1. or G bases QuikChange Site Directed Mutagenesis Kit 5 Additional Primer Considerations The mutagenesis protocol uses 125 ng of each oligonucleotide primer To convert nanograms to picomoles of oligo use the following equation ng of oligo X pmoles of oligo x1000 330 x of bases in oligo For example for 125 ng of a 25 mer 125 ng of oligo 330 x 25 bases x1000 15 pmole Primers need not be 5 phosphorylated but must be purified either by fast polynucleotide liquid chromatography FPLC or by polyacrylamide gel electrophoresis PAGE Failure to purify the primers results in a significant decrease in mutation efficiency It is important to keep primer concentration in excess Stratagene suggests varying the amount of template while keeping the concentration of the primer constantly in excess QuikChange Site Directed Mutagenesis Kit PROTOCOL Mutant Strand Synthesis Reaction Thermal Cycling Notes Ensure that the plasmid DNA template is isolated from a dam E coli strain The majority of the commonly used E coli strains are damt Plasmid DNA isolated from dam strains e g JM110 and SCS110 is not suitable To maximize temperature cycling performance Stratagene strongly recommends using thin walled tubes which ensure ideal contact with the temperature cycler s heat blocks The following protocols were optimized using thin walled tubes 1 Synthesize two complimentary oligon2. 023 0448 Switzerland 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 563 080 0800 563 082 0800 563 081 United Kingdom 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 917 3282 0800 917 3283 0800 917 3281 All Other Countries Please contact your local distributor A complete list of distributors is available at www stratagene com QuikChange Site Directed Mutagenesis Kit CONTENTS Materials Provided MON 1 Storage Gn CH OD TERR TTL DIN DLE 1 Additional Materials Required ecce eee eee e eerte ee ee ee sette sten aestas eee to setae s snae seen sese sese tasas eaa 1 Int oduclo osque I deris tuni testate RIA n pretike vx vb Ge enn o pe Ssn Un poke ut level ed P i be EIN UE 2 QuikChange Mutagenesis Control 4 ee ee eee ee esee ee ertet testen aestas ette etna esten sese ses toss seen a ee 4 Mutagenic Primer LER EE LT 5 Primer Design Guide lines eon nee e Eee e defen mei eed efle na 5 Additional Primer Considerations esses eene enne entente nnns 6 j 7 Mutant Strand Synthesis Reaction Thermal Cycling eene 7 Dpn I Digestion of the Amplification Products eeeseseeeeeeeeeeen rennen 9 Transformation of XL1 Blue Supercompetent Cells eene 9 Transformation Guidelines a cire ic erae eetobos un tbet is encie epa ein on per N ra Rene UE penR
3. A 1985 Proc Natl Acad Sci U S A 82 2 488 92 Sugimoto M Esaki N Tanaka H and Soda K 1989 Anal Biochem 179 2 309 11 Taylor J W Ott J and Eckstein F 1985 Nucleic Acids Res 13 24 8765 85 Vandeyar M A Weiner M P Hutton C J and Batt C A 1988 Gene 65 1 129 33 Papworth C Bauer J C Braman J and Wright D A 1996 Strategies 9 3 3 4 Nelson M and McClelland M 1992 Methods Enzymol 216 279 303 MSDS INFORMATION pBluescript PfuTurbo QuikChange and SURE are registered trademarks of Stratagene in the United States pWhitescript is a trademark of Stratagene Triton is a registered trademark of Rohm and Haas Co The Material Safety Data Sheet MSDS information for Stratagene products is provided on Stratagene s website at http www stratagene com MSDS Simply enter the catalog number to retrieve any associated MSDS s in a print ready format MSDS documents are not included with product shipments 14 QuikChange Site Directed Mutagenesis Kit STRATAGENE QuikChange Site Directed Mutagenesis Kit Catalog 200518 and 200519 QUICK REFERENCE PROTOCOL Prepare the control and sample reaction s as indicated below Note Stratagene recommends setting up a series of sample reactions using various concentrations ranging from 5 to 50 ng of dsDNA template e g 5 10 20 and 50 ng of dsDNA template Control Reaction Sample Reaction 5 pl of 10x reactio
4. Patent Nos 5 789 166 5 932 419 6 391 548 and patents pending U S Patent Nos 7 045 328 5 545 552 5 866 395 5 948 663 6 183 997 6 444 428 6 489 150 6 734 293 and patents pending PfuTurbo DNA polymerase has 6 fold higher fidelity in DNA synthesis than Tag DNA polymerase QuikChange Site Directed Mutagenesis Kit Step 1 Plasmid Preparation Step 2 Temperature Cycling Step 3 Digestion Step 4 Transformation Day V Y V FiGuRE 1 Overview of the QuikChange site directed mutagenesis method Mutagenic primers Mutated plasmid contains nicked circular strands Gene in plasmid with target site for mutation Denature the plasmid and anneal the oligonucleotide primers 2 containing the desired mutation X Using the nonstrand displacing action of PfuTurbo DNA polymerase extend and incorporate the mutagenic primers resulting in nicked circular strands Digest the methylated nonmutated parental DNA template with Dpn Transform the circular nicked dsDNA into XL1 Blue supercompetent cells After transformation the XL1 Blue supercompetent cells repair the nicks in the mutated plasmid LEGEND Parental DNA plasmid A Mutagenic primer Mutated DNA plasmid QuikChange Site Directed Mutagenesis Kit QUIKCHANGE MUTAGENESIS CONTROL The pWhitescript 4 5 kb control plasmid is used to test the efficiency of mut
5. QuikChange Site Directed Mutagenesis Kit INSTRUCTION MANUAL Catalog 200518 30 reactions and 200519 10 reactions Revision A For In Vitro Use Only 200518 12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Stratagene Stratagene shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product ORDERING INFORMATION AND TECHNICAL SERVICES United States and Canada Stratagene 11011 North Torrey Pines Road La Jolla CA 92037 Telephone 858 373 6300 Order Toll Free 800 424 5444 Technical Services 800 894 1304 Internet tech_services stratagene com World Wide Web www stratagene com Stratagene European Contacts Location Telephone Fax Technical Services Austria 0800 292 499 0800 292 496 0800 292 498 Belgium 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 15775 0800 15740 0800 15720 France 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 919 288 0800 919 287 0800 919 289 Germany 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 182 8232 0800 182 8231 0800 182 8234 Netherlands 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 023 0446 31 0 20 312 5700 0800
6. U DN POHRE QUERER UK EE fe ER 11 Storage Conditions 4 2 ee et e eene he teste nete Rh 11 Aliquoting Cells 55 2 t oett eee te ee ete e e e ege stats 11 Use of 14 ml BD Falcon Polypropylene Round Bottom Tubes ees 11 Lenegth ot the Heat Pulse ns rnain a EE fene Eta se ie dee eepee 11 Preparing the Agar Plates for Color Screening eee 11 Troubl sh otihg seesi 12 Preparation of Media and Reagent sssccsssccsssscssssscssssccsssssssssssssecssssssssesssssescsssssssssssssssssssasees 13 EI MDC 14 Dini m N 14 MSDS Informatlonh 2 ecco eo tei o eov oae ae eU xe eev se oe en oe o ero Vae Un ee oroare eaeo se oae aeuo e ove ee Uv ae aeuo deve Oo ne eii 14 QuikChange Site Directed Mutagenesis Kit MATERIALS PROVIDED Quantity Catalog 2005187 Catalog 200519 Materials provided 30 reactions 10 reactions PfuTurbo DNA polymerase 2 5 U ul 80 U 25 U 10x reaction buffer 500 ul 500 ul Dpn restriction enzyme 10 U l 300 U 100U Oligonucleotide control primer 1 34 mer 100 ng tl 750 ng 750 ng 5 CCATGA TTA CGC CAA GCG CGC AAT TAA CCC TCA C 3 Oligonucleotide control primer 2 34 mer 100 ng tl 750 ng 750 ng 5 GTG AGG GTT AAT TGC GCG CTT GGC GTA ATC ATG G 3 pWhitescript 4 5 kb control plasmid 5 ng ul 50 ng 50 ng dNTP mixte 30 ul 10 pl XL1 Blue s
7. agenesis Method Segment Cycles Temperature Time 1 95 C 30 seconds 2 12 18 95 C 30 seconds 55 C minute 68 C 1 minute kb of plasmid length For example a 5 kb plasmid requires 5 minutes at 68 C per cycle 5 Cycle each reaction using the cycling parameters outlined in Table I For the control reaction use a 5 minute extension time and run the reaction for 18 cycles 6 Adjust segment 2 of the cycling parameters in accordance with the type of mutation desired see the following table Type of mutation desired Number of cycles Point mutations 12 Single amino acid changes 16 Multiple amino acid deletions or insertions 18 7 Following temperature cycling place the reaction on ice for 2 minutes to cool the reaction to lt 37 C Note If desired amplification may be checked by electrophoresis of 10 ul of the product on a 196 agarose gel A band may or may not be visualized at this stage In either case proceed with Dpn I digestion and transformation QuikChange Site Directed Mutagenesis Kit Dpn Digestion of the Amplification Products Note Itis important to insert the pipet tip below the mineral oil overlay when adding the Dpn I restriction enzyme to the reaction tubes during the digestion step or when transferring the 1 ul of Dpn I treated DNA to the transformation reaction Stratagene suggests using specialized aerosol resistant pipet tips which are small and poin
8. all variations in temperature and must be stored at the bottom of a 80 C freezer Transferring tubes from one freezer to another may result in a loss of efficiency The XL1 Blue supercompetent cells should be placed at 80 C directly from the dry ice shipping container When aliquoting keep the XL1 Blue supercompetent cells on ice at all times It is essential that the BD Falcon polypropylene tubes are placed on ice before the cells are thawed and that the cells are aliquoted directly into the prechilled tubes Use of 14 ml BD Falcon Polypropylene Round Bottom Tubes It is important that 14 ml BD Falcon polypropylene round bottom tubes BD Biosciences Catalog 352059 are used for the transformation protocol because the duration of the heat pulse step is critical and has been optimized for the thickness and shape of these tubes Length of the Heat Pulse There is a defined window of highest efficiency for the XL1 Blue supercompetent cells resulting from the heat pulse in step 3 of the transformation protocol Optimal efficiencies are observed when cells are heat pulsed for 45 seconds Heat pulsing for at least 45 seconds is recommended to allow for slight variations in the length of incubation Efficiencies decrease sharply when pulsing for 30 seconds or for gt 45 seconds Preparing the Agar Plates for Color Screening To prepare the LB agar plates for blue white color screening add 80 ug ml of 5 bromo 4 chloro 3 indolyl B D gala
9. ant plasmid generation using the QuikChange site directed mutagenesis kit The pWhitescript 4 5 kb control plasmid contains a stop codon TAA at the position where a glutamine codon CAA would normally appear in the B galactosidase gene of the pBluescript II SK phagemid corresponding to amino acid 9 of the protein XL1 Blue supercompetent cells transformed with this control plasmid appear white on LB ampicillin agar plates see Preparation of Media and Reagents containing IPTG and X gal because p galactosidase activity has been obliterated The oligonucleotide control primers create a point mutation on the pWhitescript 4 5 kb control plasmid that reverts the T residue of the stop codon TAA at amino acid 9 of the B galactosidase gene to a C residue to produce the glutamine codon CAA found in the wild type sequence Following transformation colonies can be screened for the B galactosidase f gal blue phenotype 4 QuikChange Site Directed Mutagenesis Kit MUTAGENIC PRIMER DESIGN Note Mutagenic primers can be designed using Stratagene s web based QuikChange Primer Design Program available online at http www stratagene com qcprimerdesign Primer Design Guidelines The mutagenic oligonucleotide primers for use in this protocol must be designed individually according to the desired mutation The following considerations should be made for designing mutagenic primers Both of the mutagenic primers must contain
10. ctopyranoside X gal 20 mM isopropyl 1 thio D D galactopyranoside IPTG and the appropriate antibiotic to the LB agar Alternatively 100 ul of 10 mM IPTG and 100 ul of 2 X gal can be spread on the LB agar plates 30 minutes prior to plating the transformations Prepare the IPTG in sterile dH O prepare the X gal in dimethylformamide DMF Do not mix the IPTG and the X gal before pipetting them onto the plates because these chemicals may precipitate QuikChange Site Directed Mutagenesis Kit 11 TROUBLESHOOTING When used according to the guidelines outlined in this instruction manual Stratagene s kit will provide a reliable means to conduct site directed mutagenesis using dsDNA templates Undoubtedly there will be variations in the base composition and length of the DNA template and in the thermal cycler that may contribute to differences in mutagenesis efficiency Stratagene provides the following guidelines for troubleshooting these variations Observation Suggestion s Low transformation efficiency or low Ensure that excess mineral oil is not transferred into the transformation reaction colony number when pipetting the Dpn I treated DNA Using the smallest pipet tips available insert the pipet tip completely below the mineral layer overlay and clear the pipet tip while submerged beneath the mineral oil overlay before collecting the sample Ensure that sufficient mutant DNA is synthesized in the reaction Increase the a
11. d 0 1 ng ul to a 50 ul aliquot of the supercompetent cells Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30 minutes 3 Heat pulse the transformation reactions for 45 seconds at 42 C and then place the reactions on ice for 2 minutes Note This heat pulse has been optimized for transformation in 14 ml BD Falcon polypropylene round bottom tubes QuikChange Site Directed Mutagenesis Kit 9 4 Add 0 5 ml of NZY broth see Preparation of Media and Reagents preheated to 42 C and incubate the transformation reactions at 37 C for 1 hour with shaking at 225 250 rpm 5 Plate the appropriate volume of each transformation reaction as indicated in the table below on agar plates containing the appropriate antibiotic for the plasmid vector For the mutagenesis and transformation controls spread cells on LB ampicillin agar plates containing 80 ug ml X gal and 20 mM IPTG see Preparing the Agar Plates for Color Screening Transformation reaction plating volumes Reaction Type Volume to Plate pWhitescript mutagenesis control 250 ul pUC18 transformation control 5 ul in 200 ul of NZY broth Sample mutagenesis 250 ul on each of two plates entire transformation reaction Place a 200 1 pool of NZY broth on the agar plate pipet the 5 ul of the transformation reaction into the pool then spread the mixture 6 Incubate the transformation plates at 37 C for gt 16 ho
12. mount of the Dpn I treated DNA used in the transformation reaction to 4 ul Visualize the DNA template on a gel to verify the quantity and quality Nicked or linearized plasmid DNA will not generate complete circular product Verify that the template DNA is at least 80 supercoiled It is not uncommon to observe low numbers of colonies especially when generating large mutations Most of the colonies that do appear however will contain mutagenized plasmid Ethanol precipitate the Dpn digested PCR product and resuspend in a decreased volume of water before transformation Low mutagenesis efficiency or low Different thermal cyclers may contribute to variations in ramping efficiencies colony number with the control Adjust the cycling parameters for the control reaction and repeat the protocol for reaction the sample reactions Ensure that supercompetent cells are stored at the bottom of a 80 C freezer immediately upon arrival see also Transformation Guidelines Verify that the agar plates were prepared correctly See Preparing the Agar Plates for Color Screening and follow the recommendations for IPTG and X Gal concentrations carefully For best visualization of the blue B gal phenotype the control plates must be incubated for at least 16 hours at 37 C Avoid multiple freeze thaw cycles for the dNTP mix Thaw the dNTP mix once prepare single use aliquots and store the aliquots at 20 C Do not subject
13. n buffer 5 ul of 10x reaction buffer 2 ul 10 ng of pWhitescript 4 5 kb control X ul 5 50 ng of dsDNA template template 5 ng ul X ul 125 ng of oligonucleotide primer 1 1 25 ul 125 ng of oligonucleotide control X ul 125 ng of oligonucleotide primer 2 primer 1 34 mer 100 ng ul 1 pl of dNTP mix 1 25 ul 125 ng of oligonucleotide control ddH O to a final volume of 50 ul primer 2 34 mer 100 ng ul 1 ul of dNTP mix ddH O to a final volume of 50 ul Then add 1 ul of PfuTurbo DNA polymerase 2 5 U l to each control and sample reaction Overlay each reaction with 30 ul of mineral oil e Cycle each reaction using the cycling parameters outlined in the following table Segment Cycles Temperature Time 1 95 C 30 seconds 2 12 18 95 C 30 seconds 55 minute 68 C 1 minute kb of plasmid length e Adjust segment 2 of the cycling parameters in accordance with the type of mutation desired see the table in step 6 of Mutant Strand Synthesis Reaction Thermal Cycling in the instruction manual Add 1 ul of the Dpn restriction enzyme 10 U l below the mineral oil overlay Gently and thoroughly mix each reaction spin down in a microcentrifuge for 1 minute and immediately incubate at 37 C for 1 hour to digest the parental supercoiled dsDNA Transform 1 ul of the Dpn treated DNA from each control and sample reaction into separate 50 ul aliquots of XL1 Blue supercompetent cells see Transforma
14. ro site directed mutagenesis is an invaluable technique for studying protein structure function relationships and gene expression and for carrying out vector modification Several approaches to this technique have been published but these methods generally require single stranded DNA ssDNA as the template and are labor intensive or technically difficult Stratagene s QuikChange Site Directed Mutagenesis Kit allows site specific mutation in virtually any double stranded plasmid thus eliminating the need for subcloning into M13 based bacteriophage vectors and for ssDNA rescue In addition the QuikChange site directed mutagenesis system requires no specialized vectors unique restriction sites or multiple transformations This rapid four step procedure generates mutants with greater than 80 efficiency The protocol is simple and uses either miniprep plasmid DNA or cesium chloride purified DNA For long 8 kb or difficult targets Stratagene offers the QuikChange XL site directed mutagenesis kit Catalog 200516 The QuikChange site directed mutagenesis kit is used to make point mutations switch amino acids and delete or insert single or multiple amino acids The QuikChange site directed mutagenesis method is performed using PfuTurbo DNA polymerase and a temperature cycler PfuTurbo DNA polymerase replicates both plasmid strands with high fidelity and without displacing the mutant oligonucleotide primers The basic procedure utili
15. such as Stratagene s SURE 2 Supercompetent Cells Catalog 200152 Note that SURE 2 competent cells are not recommended for use with mutagenized plasmids greater than 10 kb in size note also that SURE 2 cells are Kan Tet and Chl and are not compatible with plasmid selection using kanamycin tetracycline or chloramphenicol resistance markers PREPARATION OF MEDIA AND REAGENTS LB Agar per Liter 10 g of NaCl 10 g of tryptone 5 g of yeast extract 20 g of agar Add deionized H O to a final volume of 1 liter Adjust pH to 7 0 with 5 N NaOH Autoclave Pour into petri dishes 25 ml 100 mm plate LB Ampicillin Agar per Liter 1 liter of LB agar autoclaved Cool to 55 C Add 10 ml of 10 mg ml filter sterilized ampicillin Pour into petri dishes 425 ml 100 mm plate NZY Broth per Liter 10 g of NZ amine casein hydrolysate 5 g of yeast extract 5 g of NaCl Add deionized H5O to a final volume of 1 liter Adjust to pH 7 5 using NaOH Autoclave Add the following filer sterilized supplements prior to use 12 5 ml of 1 M MgCl 12 5 ml of 1 M MgSO 20 ml of 20 w v glucose or 10 ml of 2 M glucose 10x Reaction Buffer 100 mM KCI 100 mM NH SO4 200 mM Tris HCl pH 8 8 20 mM MgSO 196 Triton X 100 1 mg ml nuclease free bovine serum albumin BSA TE Buffer 10 mM Tris HCl pH 7 5 1 mM EDTA QuikChange Site Directed Mutagenesis Kit REFERENCES ENDNOTES Kunkel T
16. ted to facilitate this process 1 Add 1 ul of the Dpn I restriction enzyme 10 U l directly to each amplification reaction below the mineral oil overlay using a small pointed pipet tip 2 Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times Spin down the reaction mixtures in a microcentrifuge for 1 minute and immediately incubate each reaction at 37 C for 1 hour to digest the parental i e the nonmutated supercoiled dsDNA Transformation of XL1 Blue Supercompetent Cells Notes Please read the Transformation Guidelines before proceeding with the transformation protocol XLI Blue cells are resistant to tetracycline If the mutagenized plasmid contains only the tet resistance marker an alternative tetracycline sensitive strain of competent cells must be used 1 Gently thaw the XL1 Blue supercompetent cells on ice For each control and sample reaction to be transformed aliquot 50 ul of the supercompetent cells to a prechilled 14 ml BD Falcon polypropylene round bottom tube 2 Transfer 1 ul of the Dpn I treated DNA from each control and sample reaction to separate aliquots of the supercompetent cells Note Carefully remove any residual mineral oil from the pipet tip before transferring the Dpn I treated DNA to the transformation reaction As an optional control verify the transformation efficiency of the XL1 Blue supercompetent cells by adding 1 ul of the pUC18 control plasmi
17. the dNTP mix to multiple freeze thaw cycles Adjust the cycling parameters for the sample reaction to overcome differences in ramping efficiencies of thermal cyclers Low mutagenesis efficiency with the Add the Dpn restriction enzyme below the mineral oil overlay in the digestion step sample reaction s and ensure proper mixing of all components in the reaction especially the Dpn I Allow sufficient time for the Dpn to completely digest the parental template repeat the digestion if too much DNA template was present Avoid multiple freeze thaw cycles for the dNTP mix Thaw the dNTP mix once prepare single use aliquots and store the aliquots at 20 C Do not subject the dNTP mix to multiple freeze thaw cycles Table continues on the following page 12 QuikChange Site Directed Mutagenesis Kit Table continues from the previous page False positives Poor quality primers can lead to false positives Radiolabel the primers and check for degradation on an acrylamide gel or resynthesize the primers mutagenic primers False priming can lead to false positives Increase the stringency of the reaction by increasing the annealing temperature to within 5 C of the melting temperature of the Unwanted deletion or recombination of plasmid DNA following mutagenesis and transformation Transform the mutagenesis reaction into competent cells that are designed to prevent recombination events
18. the desired mutation and anneal to the same sequence on opposite strands of the plasmid Primers should be between 25 and 45 bases in length with a melting temperature Tm of 278 C Primers longer than 45 bases may be used but using longer primers increases the likelihood of secondary structure formation which may affect the efficiency of the mutagenesis reaction The following formula is commonly used for estimating the T of primers T 81 5 0 41 GC 675 N mismatch For calculating Tn e Nis the primer length in bases e values for GC and mismatch are whole numbers For calculating T for primers intended to introduce insertions or deletions use this modified version of the above formula T 81 5 0 41 GC 675 N where N does not include the bases which are being inserted or deleted Note When using primer design software for QuikChange site directed mutagenesis applications be aware that the Tm calculated by the primer design software may differ from the T value calculated using the formula presented above Stratagene recommends verifying primer T s using the formula above or by using the QuikChange T calculator available online at http www stratagene com The desired mutation deletion or insertion should be in the middle of the primer with 10 15 bases of correct sequence on both sides The primers optimally should have a minimum GC content of 40 and should terminate in one or more C
19. tion of XL1 Blue Supercompetent Cells in the instruction manual
20. ucleotides containing the desired mutation flanked by unmodified nucleotide sequence Purify these oligonucleotide primers prior to use in the following steps see Mutagenic Primer Design 2 Prepare the control reaction as indicated below 5 ul of 10x reaction buffer see Preparation of Media and Reagents 2 ul 10 ng of pWhitescript 4 5 kb control plasmid 5 ng ul 1 25 ul 125 ng of oligonucleotide control primer 1 34 mer 100 ng ul 1 25 ul 125 ng of oligonucleotide control primer 2 34 mer 100 ng ul 1 ul of dNTP mix 39 5 ul of double distilled water ddH O to a final volume of 50 ul Then add 1 ul of PfuTurbo DNA polymerase 2 5 U l 3 Prepare the sample reaction s as indicated below Note Stratagene recommends setting up a series of sample reactions using various concentrations of dsDNA template ranging from 5 to 50 ng e g 5 10 20 and 50 ng of dsDNA template while keeping the primer concentration constant 5 pl of 10x reaction buffer X wl 5 50 ng of dsDNA template X wl 125 ng of oligonucleotide primer 1 X wl 125 ng of oligonucleotide primer 2 1 ul of dNTP mix ddH O to a final volume of 50 ul Then add 1 ul of PfuTurbo DNA polymerase 2 5 U l QuikChange Site Directed Mutagenesis Kit 7 4 If the thermal cycler to be used does not have a hot top assembly overlay each reaction with 30 pl of mineral oil TABLE I Cycling Parameters for the QuikChange Site Directed Mut
21. upercompetent cells blue tubes 8 x 200 ul 3 x 200 ul pUC18 control plasmid 0 1 ng pl in TE buffer 10 ul 10 ul a The QuikChange Site Directed Mutagenesis Kit Catalog 200518 contains enough reagents for 30 total reactions which includes 5 control reactions The QuikChange Site Directed Mutagenesis Kit Catalog 200519 contains enough reagents for 10 total reactions which includes 5 control reactions o See Preparation of Media and Reagents Thaw the dNTP mix once prepare single use aliquots and store the aliquots at 20 C Do not subject the dNTP mix to multiple freeze thaw cycles The composition of the dNTP mix is proprietary This reagent has been optimized for the QuikChange site directed mutagenesis protocols and has been qualified for use in conjunction with the other kit components Do not substitute with a o dNTP mixes provided with other Stratagene kits Genotype recAl endAl gyrA96 thi 1 hsdR17 supE44 relA1 lac F proAB lacl ZAM15 Tn10 Tet STORAGE CONDITIONS XL1 Blue Supercompetent Cells and pUC18 Control Plasmid 80 C All Other Components 20 C ADDITIONAL MATERIALS REQUIRED 14 ml BD Falcon polypropylene round bottom tubes BD Biosciences Catalog 352059 5 Bromo 4 chloro 3 indolyl B D galactopyranoside X gal Isopropyl 1 thio B D galactopyranoside IPTG Revision A Copyright 2006 by Stratagene QuikChange Site Directed Mutagenesis Kit 1 INTRODUCTION In vit
22. urs Expected Results for the Control Transformations The expected colony number from the transformation of the pWhitescript control mutagenesis reaction is between 50 and 800 colonies Greater than 80 of the colonies should contain the mutation and appear as blue colonies on agar plates containing IPTG and X gal Note The mutagenesis efficiency ME for the pWhitescript 4 5 kb control plasmid is calculated by the following formula ME Number of blue colony forming units cfu x 100 Total number of colony forming units cfu If transformation of the pUC18 control plasmid was performed gt 250 colonies should be observed transformation efficiency 210 cfu ug with gt 98 of the colonies having the blue phenotype Expected Results for Sample Transformations The expected colony number is between 10 and 1000 colonies depending upon the base composition and length of the DNA template employed For suggestions on increasing colony number see Troubleshooting The insert of interest should be sequenced to verify that selected clones contain the desired mutation s QuikChange Site Directed Mutagenesis Kit TRANSFORMATION GUIDELINES It is important to store the XL1 Blue supercompetent cells at 80 C to prevent a loss of efficiency For best results please follow the directions outlined in the following sections Storage Conditions Aliquoting Cells The XL1 Blue supercompetent cells are very sensitive to even sm
23. zes a supercoiled double stranded DNA dsDNA vector with an insert of interest and two synthetic oligonucleotide primers containing the desired mutation see Figure 1 The oligonucleotide primers each complementary to opposite strands of the vector are extended during temperature cycling by PfuTurbo DNA polymerase Incorporation of the oligonucleotide primers generates a mutated plasmid containing staggered nicks Following temperature cycling the product is treated with Dpn I The Dpn I endonuclease target sequence 5 Gm ATC 3 is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template and to select for mutation containing synthesized DNA DNA isolated from almost all E coli strains is dam methylated and therefore susceptible to Dpn I digestion The nicked vector DNA containing the desired mutations is then transformed into XL1 Blue supercompetent cells The small amount of starting DNA template required to perform this method the high fidelity of the PfuTurbo DNA polymerase and the low number of thermal cycles all contribute to the high mutation efficiency and decreased potential for generating random mutations during the reaction Note While plasmid DNA isolated from almost all of the commonly used E coli strains dam is methylated and is a suitable template for mutagenesis plasmid DNA isolated from the exceptional dam E coli strains including JM110 and SCS110 is not suitable U S
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