Taq DNA Polymerase

For general laboratory use. Not for usein diagnotic procedures. FOR IN VITRO USE ONLY.Taq DNA PolymeraseFrom Thermus aquaticus BM, recombinant (E. coli)Deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.75 U/lCat. No. 11 146 165 001100 UCat. No. 11 146 173 001500 UCat. No. 11 418 432 0014 × 250 UCat. No. 11 596 594 00110 × 250 UCat. No. 11 435 094 001 20 × 250 U Version Nov. 2005Store at 15 to 25°C1. What this Product DoesNumber of ReactionsIf 1.25 U are used per 50 l reaction, Taq DNA Polymerase is designedfor:• approx. 80 reactions (Cat. No. 11 146 165 001)• approx. 400 reactions (Cat. No. 11 146 173 001)• approx. 800 reactions (Cat. No. 11 418 432 001)• approx. 2000 reactions (Cat. No. 11 596 594 001)• approx. 4000 reactions (Cat. No. 11 435 094 001)ContentsLabelTaq DNA Polymerase(5 U/l)PCR reactionbuffer withMgCl 2 ,10 ×conc.Contents• 20 l (100 U pack size)• 100 l (500 U pack size)• 4 × 50 l (4 x 250 U pack size)• 10 × 50 l (10 x 250 U pack size)• 20 × 50 l (20 x 250 U pack size)Enzyme storage buffer: 20 mM Tris-HCl, 1 mM DTT,0.1 mM EDTA, 0.1 M KCl, 0.5% Nonidet P40 (v/v),0.5% Tween 20 (v/v), 50% glycerol(v/v), pH 8.0 (4°C)• 1 ml (100 U pack size)• 3 × 1 ml (500 U pack size)• 6 × 1 ml (4 x 250 U pack size)• 15 × 1 ml (10 x 250 U pack size)• 30 × 1 ml (20 x 250 U pack size)Buffer composition: 100 mM Tris-HCl, 15 mMMgCl 2 , 500 mM KCl, pH 8.3 (20°C)Storage and StabilityThe undiluted solutions are stable when stored at –15 to –25°Cthrough the control date printed on the label.Additional Equipment and Reagents Required• Template DNA, gene-specific PCR primer pair• dNTPs, PCR Grade*• Water, PCR Grade*• Thermal block cycler (e.g., Applied Biosystems GeneAmp PCR System9600)• 0.2 ml thin-walled PCR tubes*• Sterile reaction tubes for preparing master mixes and dilutionsApplication• Polymerase Chain Reaction (PCR): Taq DNA Polymerase activity isstable during prolonged incubation at high temperatures (95°C)and can therefore be used to amplify DNA fragments by PCR.• DNA labeling reactions (4, 5)• Sequencing / cycle sequencing (4, 6)Enzyme PropertiesVolume ActivityOptimal Enzyme Concentration2. How To Use this Product2.1 Before You Begin5 U/lVaries between 0.5 and 2.5 U per 50 lreactionStandard Enzyme Concentration1.25 U per 50 l reactionOptimal pHAround 9 (adjusted at 20°C)Optimal Elongation TemperatureAround 72°COptimal Mg 2+ Concentration Varies between 1.5 and 5 mM (asMgCl 2 )Standard Mg 2+ Concentration 1.5 mM (as MgCl 2 ) when used with200 M of each dNTPSize of PCR Products Enzyme optimally amplifies up to 3 kbproducts. (PCR is possible up to 10 kb,but yield diminishes as DNA fragmentlength increases.)PCR CloningT/A-cloning (Enzyme adds a single,overhanging A.)Incorporation of ModifiedNucleotidesThermostabilityEnzyme accepts modified nucleotideslike radiolabeled nucleotides, DIGdUTP,biotin-dUTP.Enzyme retains over 80% activity after30 cycles (1 min 95°C, 1 min 37°C,3 min 72°C).General considerationsThe optimal conditions (incubation times and temperatures, concentrationof enzyme, template DNA, Mg 2+ ) vary from system to systemand must be determined for each individual experimental system (7).At the very least, you should titrate the Mg 2+ concentration and theamount of enzyme used per assay to ensure optimal efficiency of DNAsynthesis.* available from Roche Applied Science1105.111809750008Roche Applied Science

As a starting point, use the following guidelines:• Optimal enzyme concentration: 0.5 – 2.5 U/50 l. A concentration of1.25 U/50 l will usually produce satisfactory results.• Optimal Mg 2+ concentration can vary between 1.5 mM and 5 mM.In most cases a Mg 2+ concentration of 1.5 mM will produce satisfactoryresults (2, 3) if you use 200 M of each dNTP.• dNTP concentration: Always use equal concentrations of all fourdNTPs. The final concentration of each dNTP should be between 50and 500 M; the most commonly used concentration is 200 M. Ifyou increase the dNTP concentration, you must also increase theMg 2+ concentration.• Template concentration: Typical concentrations are 10 ng - 250 nghuman genomic DNA and 0.1 ng - 15 ng plasmid DNA.• The optimal buffer for the template DNA is either sterile double-distilledwater or 5 - 10 mM Tris (pH 7 - 8).N Do not dissolve the template in TE buffer because EDTA chelatesMg 2+ .2.2 Preparation of Reaction MixesFor multiple reactions, we recommend that you prepare two reactionmixes. This eliminates the need for a hot start and keeps the enzymefrom interacting with primers and template during preparation of thereaction mixes. If you are setting up multiple reactions, we also recommendpreparing a Master Mix that contains all reaction componentsthat are present in each reaction. The volume of each Master Mix typicallyshould be 110% of the volume needed for all the samples. (Forexample to prepare Master Mix 2 below for 10 reactions, make 275 lof the mix.) (The extra volume allows for losses during pipetting.)Preparation of Master Mix 1• Thaw the reagents and store on ice.• Briefly vortex and centrifuge all reagents before setting up thereactions.Prepare a 10× conc. solution of each respective primer.L If you are using e.g. the final concentration of 0.5 M for eachprimer, the 10× conc. solution would contain a 5 M concentrationof the respective primer.To a sterile reaction tube on ice, add the components in theorder listed below: (For each 50 l reaction)Component Volume Final conc.Water, PCR Grade to make a finalvol. of 25 lPCR Grade Nucleotide Mix(10 mM of each dNTP)Preparation of Master Mix 21 l 200 M (of eachdNTP)Downstream primer 5 l 0.1 – 0.6 MUpstream Primer 5 l 0.1 – 0.6 MTemplate DNA variable 0.1 – 250 ngFinal volume25 lMix and centrifuge briefly.• Thaw the reagents and store on ice.• Briefly vortex and centrifuge all reagents before setting up thereactions.To a sterile reaction tube on ice, add the components in theorder listed below: (For each 50 l reaction)Component Volume Final conc.Water, PCR Grade19.75 lPCR reaction buffer, 10× 5 l 1×(1.5 mM MgCl 2 )Taq DNA Polymerase (5 U/l) 0.25 l 1.25 U/reactionFinal volume25 lMix and centrifuge briefly.2.3 PCR• For each reaction, combine 25 l Master Mix 1 and 25 l MasterMix 2 in a thin-walled PCR tube on ice.• Gently vortex the mixture to produce a homogeneous reaction,then centrifuge briefly to collect the solution at the bottom ofthe tube.N Start thermal cycling immediately. Do not store completereaction mixes on ice.Place your samples in a thermal block cycler and use either ofthe thermal profiles below to perform PCR.• Thermal Profile A: fixed extension timeCycles Time TempInitial Denaturation 1 2 min 94°CDenaturationAnnealingElongation25 – 30 15 s - 30 s30 s - 60 s45 s – 3 min94°C55 to 65°C72 or 68°CFinal Elongation 1 7 min 72 or 68°CCooling indefinitely 4°C• Thermal Profile B: gradually increasing extension time (Thisprocedure ensures a higher yield of amplification products.)Cycles Time TempInitial Denaturation 1 2 min 94°CDenaturationAnnealingElongationDenaturationAnnealingElongation10 15 s - 30 s30 s - 60 s45s – 3 min94°C55 to 65°C72 or 68°C15 – 20 15 s - 30 s 94°C30 s55 to 65°C45 s – 3 min + 5 s 72 or 68°Ccycle elongation foreach succ. cycle aFinal Elongation 1 7 min 72 or 68°CCooling indefinitely 4°CaFor example, cycle no. 11 is 5 s longer than cycle 10, cycle no. 12 is 10 slonger than cycle 10, cycle no. 13 is 15 s longer than cycle 10, etc.L The denaturation temperature can vary between 92°C and 95°C. Thestandard denaturation temperature is 94°C.Optimal annealing temperature depends on the melting temperature ofthe primers and on the experimental system.For PCR products up to 1 kb, elongation temperature should be around72°C; for PCR products larger than 1 kb, elongation temperature shouldbe around 68°C.After cycling, if the samples are not used immediately, storethem frozen for later use.L For best results, do the following:• Check the PCR product on an agarose gel for size and specificity.Use an appropriate size marker.• Purify the PCR product with the High Pure PCR Product PurificationKit* (e.g., before performing nested PCR).2.4 DIG DNA LabelingDigoxigenin 11-dUTP* is incorporated into DNA by Taq DNA Polymerase.Please refer to Roche Applied Science DIG Kits, DIG Product SelectionGuide or DIG Manuals for detailled protocols. For direct access pleasevisit http://www.roche-applied-science.com/DIG2Roche Applied Science

3. TroubleshootingPossible CauseLittle or no Difficult templatePCR product e.g., GC-rich templatesMultiplebands orbackgroundsmearDNA template problemsEnzyme concentrationtoo lowMgCl 2 concentrationtoo lowCycle conditions notoptimalPrimer design notoptimalPrimer concentrationnot optimalPrimer quality orstorage problemsFormation of primerdimersAnnealing temperaturetoo lowPrimer design orconcentration notoptimalDifficult template(e.g., GC- rich template)DNA templateproblemsRecommendation• Perform PCR with GC-RICHPCR System*.• Add DMSO (final concentration,8%) and reduce enzymeconcentration (e.g. use as littleas 0.5 U per reaction).Check quality and concentrationof template:• Analyze an aliquot on an agarosegel to check for possibledegradation.• Test the template with anestablished primer pair or PCRsystem.• Check or repeat template purification.• Increase enzyme concentrationto 2 U Taq DNA Polymeraseper 50 l reaction.• If necessary, increase theamount of polymerase in 0.5 Usteps.Increase the MgCl 2 concentrationin 0.25 mM steps. (The minimalacceptable concentration is1.5 mM MgCl 2 .)• Decrease annealing temperature.• Increase cycle number.• Make sure that the final elongationstep is included in theprogram.Design alternative primers.• Both primers must have thesame concentration.• Titrate primer concentration(0.1 – 0.6 M).• If you use an establishedprimer pair, check performancein an established PCR system(e.g. with a control template).• Make sure that the primers arenot degraded.• Always store primers at –15 to–25°C.• Use two Master Mixes, asdirected in the protocol above.• Use FastStart Taq DNA Polymerase*instead of Taq DNAPolymerase.Increase annealing temperature(Longer primers have higherannealing temperatures).• Review primer design.• Titrate primer concentration(0.1 – 0.6 M).• Both primers must have thesame concentration.• Perform nested PCR withnested primers.Perform PCR with GC-RICH PCRSystem*.Use serial dilution of template.3PCR productsin nenationCarryover contamigativecontrolexperimentsProblemsspecific toRT-PCRPossible CauseNo product, additionalbands, backgroundsmearRecommendation• Replace all reagents, especiallywater.• Use aerosol-resistant pipettetips.• Set up PCR reactions in an areaseparate from that used forPCR product analysis.• To eliminate carryover contaminants:Use dUTP* (600 M)instead of dTTP (200 M) andthermolabile UNG* (1 U/50 lreaction); also, increase Mg 2+concentration (to a maximumof 4 mM) to compensate forhigher dNTP conc.• The volume of cDNA template(RT-reaction) should notexceed 10% of the final volumeof the PCR reaction.• Follow troubleshooting tipsabove.• Increase MgCl 2 in 0.25 mMsteps.4. Additional Information on this ProductProduct DescriptionTaq DNA Polymerase (1, 2) is a highly processive 5’→3’ DNA polymerasethat lacks 3’→5’ exonuclease activity (3). It is a single polypeptidechain with a molecular weight of approx. 95 kD.Taq DNA Polymerase was originally isolated from the thermophiliceubacterium Thermus aquaticus BM, a strain lacking Taq I restrictionendonuclease. The enzyme preparation obtained from E. coli is free ofnonspecific endo- or exonucleases.Unit DefinitionOne unit Taq DNA polymerase is defined as the amount of enzymethat incorporates 10 nmol of total deoxyribonucleosidetriphosphatesinto acid precipitable DNA within 30 min at 75°C under the assay conditionsgiven above.Unit AssayIncubation buffer: 67 mM Tris/HCl; pH 8.3/25°C, 5 mM MgCl 2 , 10mM Mercaptoethanol, 0.2% Polydocanol, 0.2 mg/ml Gelatine, 0.2 mMeach dATP, dGTP, dTTP and 0.1 mM dCTP.Incubation procedure: M13mp9ss, M13 primer (17mer) and 1 Ci(α- 32 P) dCTP are incubated with suitable dilutions of Taq DNA polymerasein 50 l incubation buffer at 65°C for 60 min. The amount ofincorporated dNTPs is determined by trichloroacetic acid precipitation.4.1 References1 Chien, A., Edgar, D. B. & Trela, J. M. (1976) Deoxyribonucleic acidpolymerase from the extreme thermophile Thermus aquaticus. J.Bacteriol. 127, 1550-1557.2 Lawyer, F. C. et al. (1989) Isolation, characterization and expressionin Escherichia coli of the DNA polymerase gene from theextreme thermophile Thermus aquaticus. J. Biol. Chem. 264,6427-6437.3 Tindall, K. R. & Kunkel, T. A. (1988) Fidelity of DNA synthesis bythe Thermus aquaticus DNA polymerase. Biochemistry 27, 6008-6013.4 Innis, M. A., et al. (1988) DNA sequencing with Thermus aquaticusDNA polymerase and direct sequencing of polymerasechain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85,9436-9440.5 Lo, Y.-M. D., Mehal, W. Z. & Fleming, K. A. (1988) Rapid productionof vector-free biotinylated probes using the polymerasechain reaction. Nucleic Acids Res. 16, 8719.6 Taq polymerase: increased enzyme versatility in DNAsequencing (1988) Applied Biosystems.Roche Applied Science

7 Erlich, H. A. (ed.) (1989) PCR Technology: Principles and Applicationfor DNA Amplification, Stockton Press, New York.8 Mesquita, P. (2003) Human MUC2 mucin gene is transcriptionallyregulated by cdx homeodomain proteins in gastrointestinalcarcinoma cell lines. J. Biol. Chem. 278: 51549-51556.9 Zhu, Y. (2002) Hemin induces neuroglobin expression in neuralcells. Blood 100: 2494-2498.4.2 Quality ControlEach lot of Taq DNA Polymerase, dNTPack is tested for contaminatingactivities as described in the following:Test Buffer10 mM Tris-HCl, 1.5 mM MgCl 2 , 50 mM KCl, pH 8.3 (20°C).Absence of EndonucleasesLambda DNA (1 g) is incubated with Taq DNA Polymerase in50 l test buffer, overlaid with paraffin oil, at 65°C for 16 h. Theamount of enzyme that shows no degradation of the lambdaDNA is stated under "Endo 1."Absence of EndonucleasesEco RI/Hind III fragments (1 g) of lambda DNA is incubated withTaq DNA Polymerase in 50 l test buffer, overlaid with paraffin oil, at65°C for 16 h. The amount of enzyme that shows no alteration of thebanding pattern is stated under "Endo 2."Absence of ”Nicking Activity”Supercoiled pBR322 DNA (1 g) is incubated with Taq DNA Polymerasein 50 l test buffer, overlaid with paraffin oil, at 65°C for 4 h.The amount of enzyme that shows no relaxation of the supercoiledDNA is stated under "Nick. Act."Absence of ExonucleasesDifferent amounts of Taq DNA Polymerase are incubated in 100 l testbuffer containing [ 3 H]-labeled DNA, overlaid with paraffin oil, at 65°Cfor 4 h. The amount of enzyme that shows no exonuclease activity isstated under "Exo."5. Supplementary Information5.1 ConventionsText ConventionsTo make information consistent and memorable, the following text conventionsare used in this package insert:Text Convention UseNumbered Instructions Steps in a process that usually occur in the order listedlabeled , ,etc.Numbered Instructionslabeled , ,etc.Asterisk *Steps in a procedure that must be performed in theorder listedDenotes a product available from Roche AppliedScienceSymbolsIn this package insert the following symbols are used to highlight importantinformation:Symbol DescriptionLNInformation Note:Additional information about the current topic or procedure.Important Note:Information critical to the success of the procedure or use of theproduct.TrademarksFASTSTART and HIGH PURE are Trademarks of Roche.5.2 Ordering InformationRoche Applied Science offers a large selection of reagents and systemsfor life science research. For a complete overview of relatedproducts and manuals, please visit and bookmark our home pagewww.roche-applied-science.com and our Special Interest Sites including:http://www.roche-applied-science.com/PCR/NucleotidesDNAPurificationAdditionalReagentsProduct Pack Size Cat. No.PCR NucleotideMix200 l2000 lNOTICE TO PURCHASER: LIMITED LICENSEA license under the non-U.S. counterparts of U.S. Patents Nos. 4,683,202,4,683,195 and 4,965,188 owned by F. Hoffmann-La Roche Ltd. (Roche), for usein research and development, has an up-front fee component and a runningroyaltycomponent. The purchase price of the product includes a limited, nontransferablelicense under the running-royalty component to use only thisamount of product to practice the Polymerase Chain Reaction (PCR) andrelated processes described in said patents solely for the research and developmentactivities of the purchaser when this product is used in conjunctionwith a thermal cycler whose use is covered by the up-front fee component.Rights to the up-front fee component must be obtained by the end user inorder to have a complete license. These rights under the up-front fee componentmay be purchased from Applied Biosystems or obtained by purchasing anauthorized thermal cycler. This product is also an authorized reagent and mayalso be used under service sublicenses from Applied Biosystems under theforegoing patents. No right to perform or offer commercial services of any kindusing PCR, including without limitation reporting the results of purchaser’sactivities for a fee or other commercial consideration, is hereby grantedexpressly, by implication or estoppel. This product is for research use only.Diagnostic uses require a separate license from Roche. Further information onpurchasing licenses to practice the PCR process may be obtained by contactingthe Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive,Foster City, California 94404, USA.Contact and SupportTo ask questions, solve problems, suggest enhancements or report newapplications, please visit our Online Technical Support Site at:www.roche-applied-science.com/support11 581 295 00111 814 362 001High Pure PCR 100 purifications 11 796 828 001Template PurificationKitHigh Pure PCRProduct PurificationKit50 purifications250 purificationsTaq DNA Polymerase,dNTPack 100 500 UU4× 250 U10 × 250 U20 ×250 UDigoxigenin-11-dUTP(alkali-labile)Digoxigenin-11-dUTP(alkali-stable)25 nmol (25 l)125 nmol (125 l)11 732 668 00111 732 676 00104 728 866 00104 728 874 00104 728 882 00104 728 904 00104 728 858 00111 573 152 91011 573 179 91025 nmol (25 l) 11 093 088 910Biotin-16-dUTP 50 nmol (50 l) 11 093 070 910Fluorescein-12- 25 nmol (25 l) 11 373 242 910dUTPWater,PCR GradeThin-walled PCRTubes25 ml03 315 932 001(25 vials of 1 ml)25 ml (1 vial of 25 ml) 03 315 959 001100 ml(4 vials of 25 ml) 03 315 843 0011000 tubes (200 l)1000 tubes (500 l)11 667 041 00111 667 050 001To call, write, fax, or email us, visit the Roche Applied Science home page,www.roche-applied-science.com, and select your home country. Countryspecificcontact information will be displayed. Use the Product Search functionto find Pack Inserts and Material Safety Data Sheets.Roche Diagnostics GmbHRoche Applied Science68298 MannheimGermany