For life science research only. Not for use in diagnostic procedures.

Taq DNA , 1 U/␮l From BM, recombinant (E. coli) Deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7 Cat. No. 11 647 679 001 250 U y Version 14 Content version: April 2016 Cat. No. 11 647 687 001 1,000 U (4 × 250 U) Store at Ϫ15 to Ϫ25°C

1. What this Product Does Properties Volume activity 1 U/␮l If 1.25 U are used per 50 ␮l reaction, Taq DNA Polymerase is designed for approximately: Optimal enzyme concentration varies between 0,5 and 2.5 U per 50 ␮ • 200 reactions (Cat. No. 11 647 679 001) l reaction • 800 reactions (Cat. No. 11 647 687 001) Standard enzyme concentration 1.25 U per 50 ␮l reaction

2+ Contents Optimal Mg concentration varies between 1 and 10 mM (as MgCl2) Label Contents 2+ Standard Mg concentration 1.5 mM (as MgCl2) when using Taq DNA Polymerase • 250 ␮l (250 U pack size) 200 ␮M dNTP each (1 U/␮l) • 4 × 250 ␮l (1,000 U pack size) Enzyme storage buffer: 20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 0.1 M KCl, 0.5% Non- 2. How To Use this Product idet P40 (v/v), 0.5% Tween 20 (v/v), 50% glyc- erol (v/v), pH 8.0 (+4°C). 2.1 Before You Begin

PCR reaction buffer •1 ml General considerations with MgCl ,10 × conc. , 500 mM 2 • 100 mM Tris-HCl, 15 mM MgCl2 The optimal conditions of Taq DNA polymerase (incubation, times and KCl, pH 8.3 (+20°C) temperatures, concentrations of the enzyme, template DNA, primers, 2+ MgCl stock solution • 1 ml Mg ) depend on the system used and have to be determined indivi- 2 dually. •25 mM MgCl 2 Especially the Mg2+ concentration and the amount of enzyme used per PCR reaction buffer •1 ml assay should be titrated for optimal efficiency of DNA synthesis. without MgCl2, 10 × • 100 mM Tris-HCl, 500 mM KCl, pH 8.3 conc. (+20°C) Optimization of the MgCl2 concentration The optimal MgCl2 concentration must be determined empirically. In Storage and Stability most cases a concentration of 1.5 mM will produce satisfactory results. 2+ The undiluted enzyme solution is stable when stored at –15 to –25°C For individual optimization of the Mg concentration a buffer without until the expiration date printed on the label. MgCl2 and a MgCl2 stock solution are supplied separately. The table below gives the volumes of the MgCl2 stock solution which ␮ Additional Equipment and Reagents Required give the designated MgCl2 concentrations when added to a 50 l PCR mixture. All other steps for preparing of the reaction mix are the same • Template DNA, gene-specific PCR primer pair as described above. • dNTPs, PCR Grade*; Water, PCR Grade*

• Thermal block cycler (e.g., Applied Biosystems GeneAmp PCR MgCl 1 mM 1.25 mM 1.5 mM 1.75 mM 2 mM 2.5 mM 5 mM System 9600) 2 • 0.2 ml thin-walled PCR tubes Volume 2 ␮l2.5 ␮l3 ␮l3.5 ␮l4 ␮l5 ␮l10 ␮l • Sterile reaction tubes for preparing master mixes and dilutions 2.2 Preparation of Reaction Mixes * available from Roche Diagnostics For multiple reactions, we recommend that you prepare two reaction Application mixes. This eliminates the need for a hot start and keeps the enzyme Polymerase Chain Reaction (PCR): Taq DNA Polymerase activity is sta- from interacting with primers and template during preparation of the ble during prolonged incubation at high temperatures (+95°C) and reaction mixes. If you are setting up multiple reactions, we also recom- can therefore be used to amplify DNA fragments by PCR. mend preparing a Master Mix that contains all reaction components that are present in each reaction. The volume of each Master Mix typi- • DNA labeling reactions (6, 7) cally should be 110% of the volume needed for all the samples. E.g., to • Sequencing/ cycle sequencing (6, 8). prepare Master Mix 2 below for 20 reactions, make 550 ␮l of the mix (the extra volume allows for losses during pipetting).

0416.116539890014 sigma-aldrich.com Preparation of Master Mix 1 » An example of a cycle profile is given for the Perkin Elmer Gene- ³ • Thaw the reagents and store on ice. Amp 9600 Thermal Cycler. • Briefly vortex and centrifuge all reagents before setting up the  When using other thermal block cyclers the cycle conditions reactions. have to be adjusted. Run 30 cycles for DNA and 35 cycles for cDNA. · Prepare a 10× conc. solution of each respective PCR primer.  If you are using, e.g., a final concentration of 0.5 ␮M for each Cycles Time Temp primer, the 10× conc. solution would contain a 5 ␮M concen- Initial Denaturation 1 2 min 94°C* tration of the respective primer. Denaturation 25 to 30 15 to 30 sec 94°C » Add the following reagents in a sterile 1.5 ml reaction tube on Annealing 30 to 60 sec 45 to 68°C1 ice, in the following order: Elongation 45 sec to 3 min2 72°C Component Volume Final conc. Final Elongation 1 7 min 72°C Water, PCR Grade to make a final Cooling indefinitely 4°C vol. up to 25 ␮l 1 PCR Grade Mix 1 ␮l200 ␮M (of each Annealing temperature depends on the melting temperature of the primers used. (10 mM of each dNTP) dNTP) 2 Elongation time depends on the length of the fragment to be amplified. Downstream primer 5 ␮l0.1 to 1 ␮M Use 45 sec for targets up to 1 kb, use 1 min for fragments up to 1.5 kb and 2 min for fragments up to 3 kb. ␮ ␮ Upstream primer 5 l0.1 to 1 M ¿ Samples can be stored at +2 to +8°C or -15 to -25°C. template DNA variable 10 to 250 ng gDNA After cycling, if the samples are not used immediately, store 0.1 to 15 ng cDNA them frozen for later use. For best results, do the following: Final volume 25 ␮l  – Check the PCR product on an agarose gel for size and spec- ᕤ Mix and centrifuge briefly. ificity. Use an appropriate size marker. – Purify the PCR product with the High Pure PCR Product Puri- fication Kit* (e.g., before performing nested PCR) Preparation of Master Mix 2 ³ • Thaw the reagents and store on ice. 2.3 DIG DNA Labeling • Briefly vortex and centrifuge all reagents before setting up the reactions. Digoxigenin 11-dUTP is incorporated into DNA by Taq DNA Poly- merase. · Add the following reagents in a 1.5 ml reaction tube on ice, in the following order: 3. Troubleshooting Component Volume Final conc.

Water, PCR Grade to make a final Possible Cause Recommendation vol. up to 25 ␮l Little or no Difficult template, • Perform PCR with GC-RICH ␮ PCR reaction buffer, 10 × 5 l PCR product e.g., GC-rich PCR System*. Taq DNA Polymerase, 0.5 to 2.5 ␮l 0.5 to 2.5 U/50 ␮l templates • Add DMSO (final concentra- (1 U/␮l) tion, 8%) and reduce enzyme concentration (e.g., use as little ␮ Final volume 25 l as 0.5 U per reaction). » Mix and centrifuge briefly. DNA template Check quality and concentration problems of template: PCR • Analyze an aliquot on an aga- ³ • For each reaction, combine 25 ␮l Master Mix 1 and 25 ␮l Mas- rose gel to check for possible ter Mix 2 in a thin-walled PCR tube on ice. degradation. • Gently vortex the mixture to produce a homogeneous reaction, • Test the template with an then centrifuge briefly to collect the solution at the bottom of established primer pair or PCR the tube. system. • Check or repeat template puri-  Start thermal cycling immediately. Do not store complete fication. reaction mixes on ice. Enzyme concentra- • Increase enzyme concentra- · Place your sample in a thermal block cycler and perform PCR. tion too low tion to 2 U Taq DNA Poly- merase per 50 ␮l reaction. • If necessary, increase the amount of polymerase in 0.5 U steps.

MgCl2 concentra- Increase the MgCl2 concentra- tion too low tion in 0.25 mM steps. (The mini- mal acceptable concentration is 1.5 mM MgCl2). Cycle conditions not • Decrease annealing tempera- optimal ture. • Increase cycle number. • Make sure that the final elon- gation step is included in the program. Primer design not Design alternative primers. optimal

2 sigma-aldrich.com Possible Cause Recommendation 4. Additional Information on this Product Primer concentra- • Both primers must have the Product Description tion not optimal same concentration. The enzyme was cloned in E. coli and is isolated to be free of • Titrate primer concentration unspecific endo- or according to the current quality (0.1 to 1 ␮M). control procedures. Taq DNA Polymerase (1, 2) is a highly processive Primer quality or • If you use an established 5’-3’ DNA polymerase that lacks 3’-5’ activity (3). It con- storage problems primer pair, check performance sists of a single polypeptide chain with a molecular weight of approxi- in an established PCR system mately 95 kDa.The enzyme exhibits highest activity at a pH of around 9 (e.g., with a control template). (adjusted at +20°C) and temperatures around +75°C. Taq DNA Poly- merase also accepts modified deoxyribonucleosidetriphosphates as • Make sure that the primers are substrates, and can be used to label DNA-fragments either with radio- not degraded. , digoxigenin (see above), fluorescein or biotin (6,7). • Always store primers at –15 to The high processivity, absence of exonuclease activity and tempera- –25°C. ture optima of Taq DNA Polymerase enable the use of this enzyme in Formation of primer • Use two Master Mixes, as DNA sequencing, especially where the resolution of secondary struc- dimers directed in the protocol above. tures plays a major role (8, 9). • Use FastStart Taq DNA Poly- merase* instead of Taq DNA Volume activity Polymerase. 1 U/␮l as determined in the assay on activated DNA.

Multiple Annealing tempera- Increase annealing temperature Origin bands or ture too low (Longer primers have higher background annealing temperatures). Taq DNA Polymerase (2, 3, 4) was originally isolated from the thermo- smear philic eubacterium Thermus aquaticus BM, a strain lacking Taq I Primer design or • Review primer design. restriction endonuclease. concentration not • Titrate primer concentration optimal (0.1 to 0.6 ␮M). Unit Assay on Activated DNA • Both primers must have the Incubation buffer: 67 mM Tris/HCl; pH 8.3 (+25°C), 5 mM MgCl2, 10 same concentration. mM Mercaptoethanol, 0.2% Polydocanol, 0.2 mg/ml Gelatine, 0.2 mM • Perform nested PCR with each dATP, dGTP, dTTP and 0.1 mM dCTP. nested primers. Incubation procedure: M13mp9ss, M13 primer (17mer) and 1 ␮Ci α32 Difficult template Perform PCR with GC-RICH PCR [ P] dCTP are incubated with suitable dilutions of Taq DNA Poly- ␮ (e.g., GC-rich tem- System*. merase in 50 l incubation buffer for 60 min at +65°C. The amount of plate) incorporated dNTPs is determined by trichloroacetic acid precipitation. DNA template Use serial dilution of template. Unit Definition problems One unit Taq DNA Polymerase is defined as the amount of enzyme PCR Carryover contami- • Replace all reagents, espe- that incorporates 10 nmol of total deoxyribonucleoside triphosphates products in nation cially water. into acid precipitable DNA within 60 min at +65°C under the assay negative • Use aerosol-resistant pipette conditions stated above. control tips. experiments • Set up PCR reactions in an area References separate from that used for 1 Baril, E. (1977) Nucleic Acids Res. 4, 2641–2653. PCR product analysis. 2 Chien, A., D. B. & Trela, J. M. (1976) Deoxyribonucleic acid poly- • To eliminate carryover contam- merase from the extreme Edgarthermophile Thermus aquaticus. inants: Use dUTP* (600 ␮M) J. Bacteriol. 127, 1550-1557. instead of dTTP (200 ␮M) and 3 Kaledin, A. S., Slyusarenko, A. G. & Gorodetskii, S. I. (1980) thermolabile UNG* (1 U/50 ␮l Biokhymia 45, 644– 651. reaction); also, increase Mg2+ 4 Lawyer, F. C. et al. (1989) Isolation, characterization and expres- concentration (to a maximum sion in of the DNA polymerase gene from the of 4 mM) to compensate for extreme Thermus aquaticus. J. Biol. Chem. 264, higher dNTP conc. 6427-6437. Problems No product, addi- • The volume of cDNA template 5 Tindall, K. R. & Kunkel, T. A. (1988) Fidelity of DNA synthesis by specific to tional bands, back- (RT-reaction) should not the Thermus aquaticus DNA polymerase. Biochemistry 27, 6008- RT-PCR ground smear exceed 10% of the final volume 6013. of the PCR reaction. 6 Innis, M. A., et al. (1988) DNA sequencing with Thermus aqua- • Follow troubleshooting tips ticus DNA polymerase and direct sequencing of polymerase above. chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 9436-9440. • Increase MgCl in 0.25 mM 2 7 Lo, Y.-M. D., Mehal, W. Z. & Fleming, K. A. (1988) Rapid produc- steps. tion of vector-free biotinylated probes using the polymerase chain reaction. Nucleic Acids Res. 16, 8719. 8 Taq polymerase: increased enzyme versatility in DNA sequencing (1988) Applied Biosystems. 9Innis, M.A. et al. (1988) DNA sequencing with Thermus aqua- ticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. U S A. 85, 9436-9440. 10 Erlich, H. A. (ed.) (1989) PCR Technology: Principles and Appli- cation for DNA Amplification, Stockton Press, New York.

3 sigma-aldrich.com Quality Control Product Pack Size Cat. No. Each lot of Taq DNA Polymerase is tested for contaminating activities described in the following: Amplifi- Taq DNA Poly- 250 U 04 738 225 001 cation merase (1 U/µl), 1,000 U 04 738 241 001 e.g., Test Buffer dNTPack Taq DNA Poly- 100 U 04 728 866 001 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3 (+20°C). merase, dNTPack 500 U 04 728 874 001 Absence of Endonucleases 4 × 250 U 04 728 882 001 ␮ ␮ 10 × 250 U 04 728 904 001 1 g lambda DNA is incubated with Taq DNA Polymerase in 50 l test 20 × 250 U 04 728 858 001 buffer for 16 hours at +37°C. The amount of enzyme showing no deg- radation of the lambda DNA is >20 U. Digoxigenin-11- 25 nmol (25 ␮l) 11 093 088 910 dUTP Absence of Nicking Activity (alkali-stable) 1 ␮g supercoiled pBR322 DNA is incubated with Taq DNA Polymerase Biotin-16-dUTP 50 nmol (50 ␮l) 11 093 070 910 in 50 ␮l test buffer for 4 hours at +37°C. The amount of enzyme show- Fluorescein-12- 25 nmol (25 ␮l) 11 373 242 910 ing no relaxation of supercoiled DNA is >20 U. dUTP Absence of Exonucleases Water, 25 ml 03 315 932 001 ␮ PCR Grade (25 × 1 ml) Different amounts of Taq DNA Polymerase are incubated in 100 l test 3 25 ml 03 315 959 001 buffer containing [ H]-labeled DNA with a paraffin oil overlay for (1 × 25 ml) 4 hours at +65°C. The amount of enzyme that shows no exonuclease 100 ml 03 315 843 001 activity is >3 U. (4 × 25 ml) DNA, MB-grade 500 mg (50 ml) 11 467 140 001 5. Supplementary Information (from fish sperm)

5.1 Conventions Regulatory Disclaimer For life science research only. Not for use in diagnostic procedures. Text Conventions To make information consistent and memorable, the following text conventions are used in this document: Trademarks Text Convention Use FASTSTART and HIGH PURE are Trademarks of Roche. All other product names and trademarks are the property of their Numbered Instructions Steps in a process that usually occur in the order listed labeled ቢ, ባ, etc. respective owners. Numbered Instructions Steps in a procedure that must be performed in the Disclaimer of License labeled ³, ·, etc. order listed For patent license limitations for individual products please refer to: Asterisk * Denotes a product available from Roche Diagnostics List of biochemical reagent products

Symbols In this document, the following symbols are used to highlight important infor- mation: Symbol Description Information Note:  Additional information about the current topic or procedure. Important Note:  Information critical to the success of the procedure or use of the product.

5.2 Changes to Previous Version • Editorial changes

5.3 Ordering Information

Product Pack Size Cat. No. Amplifi- Taq DNA Poly- 250 U 04 738 225 001 cation merase (1 U/µl), 1,000 U 04 738 241 001 e.g., dNTPack

Taq DNA Poly- 100 U 04 728 866 001 Contact and Support merase, dNTPack 500 U 04 728 874 001 4 × 250 U 04 728 882 001 To ask questions, solve problems, suggest enhancements and report new 10 × 250 U 04 728 904 001 applications, please visit our Online Technical Support Site. 20 × 250 U 04 728 858 001 To call, write, fax, or email us, visit sigma-aldrich.com, and select your home DNA High Pure PCR 100 purifications 11 796 828 001 country. Country-specific contact information will be displayed. Purifica- Template tion Purification Kit High Pure PCR 50 purifications 11 732 668 001 Product Purifica- 250 purifications 11 732 676 001 tion Kit Addi- Digoxigenin-11- 25 nmol (25 ␮l) 11 573 152 910 tional dUTP 125 nmol (125 ␮l) 11 573 179 910 Roche Diagnostics GmbH Reagents (alkali-labile) Sandhofer Strasse 116 68305 Mannheim Germany