For general laboratory use. Not for use in diagnotic procedures. FOR IN VITRO USE ONLY.

Taq DNA Polymerase

From Thermus aquaticus BM, recombinant (E. coli) Deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.U/l
Cat. No. Cat. No. Cat. No. Cat. No. Cat. No. 094 001

100 U 500 U U U U

Version Nov. 2005

Store at 15 to 25C

What this Product Does
Number of Reactions If 1.25 U are used per 50 l reaction, Taq DNA Polymerase is designed for: approx. 80 reactions (Cat. No. 165 001) approx. 400 reactions (Cat. No. 173 001) approx. 800 reactions (Cat. No. 432 001) approx. 2000 reactions (Cat. No. 594 001) approx. 4000 reactions (Cat. No. 094 001) Contents Label Taq DNA Polymerase (5 U/l) Contents 20 l (100 U pack size) 100 l (500 U pack size) l (4 x 250 U pack size) l (10 x 250 U pack size) 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 (4C) 1 ml (100 U pack size) ml (500 U pack size) ml (4 x 250 U pack size) ml (10 x 250 U pack size) ml (20 x 250 U pack size) Buffer composition: 100 mM Tris-HCl, 15 mM MgCl2, 500 mM KCl, pH 8.3 (20C)
Application Polymerase Chain Reaction (PCR): Taq DNA Polymerase activity is stable during prolonged incubation at high temperatures (95C) and can therefore be used to amplify DNA fragments by PCR. DNA labeling reactions (4, 5) Sequencing / cycle sequencing (4, 6) Enzyme Properties Volume Activity Optimal Enzyme Concentration 5 U/l Varies between 0.5 and 2.5 U per 50 l reaction
Standard Enzyme Concentra- 1.25 U per 50 l reaction tion Optimal pH Around 9 (adjusted at 20C) Optimal Elongation Tempera- Around 72C ture Optimal Mg2+ Concentration Varies between 1.5 and 5 mM (as MgCl2) Standard Mg2+ Concentration 1.5 mM (as MgCl2) when used with 200 M of each dNTP Size of PCR Products Enzyme optimally amplifies up to 3 kb products. (PCR is possible up to 10 kb, but yield diminishes as DNA fragment length increases.) T/A-cloning (Enzyme adds a single, overhanging A.) Enzyme accepts modified nucleotides like radiolabeled nucleotides, DIGdUTP, biotin-dUTP. Enzyme retains over 80% activity after 30 cycles (1 min 95C, 1 min 37C, 3 min 72C).
PCR reaction buffer with MgCl2 ,10 conc.
PCR Cloning Incorporation of Modified Nucleotides Thermostability
Storage and Stability The undiluted solutions are stable when stored at 15 to 25C through 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 System 9600) 0.2 ml thin-walled PCR tubes* Sterile reaction tubes for preparing master mixes and dilutions

How To Use this Product

Before You Begin
General considerations The optimal conditions (incubation times and temperatures, concentration of enzyme, template DNA, Mg2+) vary from system to system and must be determined for each individual experimental system (7). At the very least, you should titrate the Mg2+ concentration and the amount of enzyme used per assay to ensure optimal efficiency of DNA synthesis.
* available from Roche Applied Science

1105.111809750008

Roche Applied Science
As a starting point, use the following guidelines: Optimal enzyme concentration: 0.5 2.5 U/50 l. A concentration of 1.25 U/50 l will usually produce satisfactory results. Optimal Mg2+ concentration can vary between 1.5 mM and 5 mM. In most cases a Mg2+ concentration of 1.5 mM will produce satisfactory results (2, 3) if you use 200 M of each dNTP. dNTP concentration: Always use equal concentrations of all four dNTPs. The final concentration of each dNTP should be between 50 and 500 M; the most commonly used concentration is 200 M. If you increase the dNTP concentration, you must also increase the Mg2+ concentration. Template concentration: Typical concentrations are 10 ng - 250 ng human genomic DNA and 0.1 ng - 15 ng plasmid DNA. The optimal buffer for the template DNA is either sterile double-distilled water or 5 - 10 mM Tris (pH 7 - 8). N Do not dissolve the template in TE buffer because EDTA chelates Mg2+. 2.2 Preparation of Reaction Mixes For multiple reactions, we recommend that you prepare two reaction mixes. This eliminates the need for a hot start and keeps the enzyme from interacting with primers and template during preparation of the reaction mixes. If you are setting up multiple reactions, we also recommend preparing a Master Mix that contains all reaction components that are present in each reaction. The volume of each Master Mix typically should be 110% of the volume needed for all the samples. (For example to prepare Master Mix 2 below for 10 reactions, make 275 l of 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 the reactions. Prepare a 10 conc. solution of each respective primer. L If you are using e.g. the final concentration of 0.5 M for each primer, the 10 conc. solution would contain a 5 M concentration of the respective primer. To a sterile reaction tube on ice, add the components in the order listed below: (For each 50 l reaction) Component Water, PCR Grade PCR Grade Nucleotide Mix (10 mM of each dNTP) Downstream primer Upstream Primer Template DNA Final volume Mix and centrifuge briefly. Volume to make a final vol. of 25 l 1 l 5 l 5 l variable 25 l 200 M (of each dNTP) 0.1 0.6 M 0.1 0.6 M 0.ng Final conc.

PCR For each reaction, combine 25 l Master Mix 1 and 25 l Master Mix 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 of the tube. N Start thermal cycling immediately. Do not store complete reaction mixes on ice.
Place your samples in a thermal block cycler and use either of the thermal profiles below to perform PCR. Thermal Profile A: fixed extension time Cycles Initial Denaturation Denaturation Annealing Elongation Final Elongation Cooling 30 Time 2 min 15 s - 30 s 30 s - 60 s 45 s 3 min 7 min Temp 94C 94C 55 to 65C 72 or 68C 72 or 68C 4C

indefinitely

Thermal Profile B: gradually increasing extension time (This procedure ensures a higher yield of amplification products.) Cycles Initial Denaturation Denaturation Annealing Elongation Denaturation Annealing Elongation Time 2 min 15 s - 30 s 30 s - 60 s 45s 3 min Temp 94C 94C 55 to 65C 72 or 68C
94C 15 s - 30 s 55 to 65C 30 s 45 s 3 min + 5 s 72 or 68C cycle elongation for each succ. cyclea 7 min 72 or 68C 4C

Final Elongation Cooling

example, cycle no. 11 is 5 s longer than cycle 10, cycle no. 12 is 10 s longer than cycle 10, cycle no. 13 is 15 s longer than cycle 10, etc. L The denaturation temperature can vary between 92C and 95C. The standard denaturation temperature is 94C. Optimal annealing temperature depends on the melting temperature of the primers and on the experimental system. For PCR products up to 1 kb, elongation temperature should be around 72C; for PCR products larger than 1 kb, elongation temperature should be around 68C.
After cycling, if the samples are not used immediately, store them 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 Purification Kit* (e.g., before performing nested PCR).
Preparation of Master Mix 2 Thaw the reagents and store on ice. Briefly vortex and centrifuge all reagents before setting up the reactions. To a sterile reaction tube on ice, add the components in the order listed below: (For each 50 l reaction) Component Water, PCR Grade PCR reaction buffer, 10 Volume 19.75 l 5 l 1 (1.5 mM MgCl2) 1.25 U/reaction Final conc.
2.4 DIG DNA Labeling Digoxigenin 11-dUTP* is incorporated into DNA by Taq DNA Polymerase. Please refer to Roche Applied Science DIG Kits, DIG Product Selection Guide or DIG Manuals for detailled protocols. For direct access please visit http://www.roche-applied-science.com/DIG
Taq DNA Polymerase (5 U/l) 0.25 l Final volume Mix and centrifuge briefly. 25 l

Troubleshooting

Possible Cause Recommendation Perform PCR with GC-RICH PCR System*. Add DMSO (final concentration, 8%) and reduce enzyme concentration (e.g. use as little as 0.5 U per reaction).

Possible Cause

Recommendation
Little or no Difficult template PCR product e.g., GC-rich templates
DNA template pro- Check quality and concentration blems of template: Analyze an aliquot on an agarose gel to check for possible degradation. Test the template with an established primer pair or PCR system. Check or repeat template purification. Enzyme concentra- Increase enzyme concentration too low tion to 2 U Taq DNA Polymerase per 50 l reaction. If necessary, increase the amount of polymerase in 0.5 U steps. MgCl2 concentration too low Increase the MgCl2 concentration in 0.25 mM steps. (The minimal acceptable concentration is 1.5 mM MgCl2.)
Carryover contami- Replace all reagents, espePCR products in ne- nation cially water. gative control Use aerosol-resistant pipette experiments tips. Set up PCR reactions in an area separate from that used for PCR product analysis. To eliminate carryover contaminants: Use dUTP* (600 M) instead of dTTP (200 M) and thermolabile UNG* (1 U/50 l reaction); also, increase Mg2+ concentration (to a maximum of 4 mM) to compensate for higher dNTP conc. Problems specific to RT-PCR No product, addi- The volume of cDNA template tional bands, back- (RT-reaction) should not ground smear exceed 10% of the final volume of the PCR reaction. Follow troubleshooting tips above. Increase MgCl2 in 0.25 mM steps.
Additional Information on this Product
Cycle conditions not Decrease annealing temperaoptimal ture. Increase cycle number. Make sure that the final elongation step is included in the program. Primer design not optimal Primer concentration not optimal Design alternative primers. Both primers must have the same concentration. Titrate primer concentration (0.1 0.6 M). If you use an established primer pair, check performance in an established PCR system (e.g. with a control template). Make sure that the primers are not degraded. Always store primers at 15 to 25C.
Product Description Taq DNA Polymerase (1, 2) is a highly processive 53 DNA polymerase that lacks 35 exonuclease activity (3). It is a single polypeptide chain with a molecular weight of approx. 95 kD. Taq DNA Polymerase was originally isolated from the thermophilic eubacterium Thermus aquaticus BM, a strain lacking Taq I restriction endonuclease. The enzyme preparation obtained from E. coli is free of nonspecific endo- or exonucleases. Unit Definition One unit Taq DNA polymerase is defined as the amount of enzyme that incorporates 10 nmol of total deoxyribonucleosidetriphosphates into acid precipitable DNA within 30 min at 75C under the assay conditions given above. Unit Assay Incubation buffer: 67 mM Tris/HCl; pH 8.3/25C, 5 mM MgCl2, 10 mM Mercaptoethanol, 0.2% Polydocanol, 0.2 mg/ml Gelatine, 0.2 mM each dATP, dGTP, dTTP and 0.1 mM dCTP. Incubation procedure: M13mp9ss, M13 primer (17mer) and 1 Ci (-32P) dCTP are incubated with suitable dilutions of Taq DNA polymerase in 50 l incubation buffer at 65C for 60 min. The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation. 4.1 References 1 Chien, A., Edgar, D. B. & Trela, J. M. (1976) Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J. Bacteriol. 127, 1550-1557. 2 Lawyer, F. C. et al. (1989) Isolation, characterization and expression in Escherichia coli of the DNA polymerase gene from the extreme thermophile Thermus aquaticus. J. Biol. Chem. 264, 6427-6437. 3 Tindall, K. R. & Kunkel, T. A. (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27, 60086013. 4 Innis, M. A., et al. (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 9436-9440. 5 Lo, Y.-M. D., Mehal, W. Z. & Fleming, K. A. (1988) Rapid production of vector-free biotinylated probes using the polymerase chain reaction. Nucleic Acids Res. 16, 8719. 6 Taq polymerase: increased enzyme versatility in DNA sequencing (1988) Applied Biosystems.

Primer quality or storage problems
Formation of primer Use two Master Mixes, as dimers directed in the protocol above. Use FastStart Taq DNA Polymerase* instead of Taq DNA Polymerase. Annealing temperaMultiple bands or ture too low background smear Primer design or concentration not optimal Increase annealing temperature (Longer primers have higher annealing temperatures). Review primer design. Titrate primer concentration (0.1 0.6 M). Both primers must have the same concentration. Perform nested PCR with nested primers.
Difficult template Perform PCR with GC-RICH PCR (e.g., GC- rich tem- System*. plate) DNA template problems Use serial dilution of template.
7 Erlich, H. A. (ed.) (1989) PCR Technology: Principles and Application for DNA Amplification, Stockton Press, New York. 8 Mesquita, P. (2003) Human MUC2 mucin gene is transcriptionally regulated by cdx homeodomain proteins in gastrointestinal carcinoma cell lines. J. Biol. Chem. 278: 51549-51556. 9 Zhu, Y. (2002) Hemin induces neuroglobin expression in neural cells. Blood 100: 2494-2498. 4.2 Quality Control
5.2 Ordering Information Roche Applied Science offers a large selection of reagents and systems for life science research. For a complete overview of related products and manuals, please visit and bookmark our home page www.roche-applied-science.com and our Special Interest Sites including: http://www.roche-applied-science.com/PCR/ Product Nucleotides PCR Nucleotide Mix Pack Size 200 l 2000 l Cat. No. 828 001
Each lot of Taq DNA Polymerase, dNTPack is tested for contaminating activities as described in the following:
Test Buffer 10 mM Tris-HCl, 1.5 mM MgCl2 , 50 mM KCl, pH 8.3 (20C). Absence of Endonucleases
DNA High Pure PCR 100 purifications Purifica- Template Purification tion Kit High Pure PCR 50 purifications Product Purifica- 250 purifications tion Kit AddiTaq DNA Poly100 U tional merase, dNTPack 500 U Reagents U U U Digoxigenin-11dUTP (alkali-labile) Digoxigenin-11dUTP (alkali-stable) Biotin-16-dUTP Fluorescein-12dUTP Water, PCR Grade 25 nmol (25 l) 125 nmol (125 l) 25 nmol (25 l) 50 nmol (50 l) 25 nmol (25 l)

088 910

Lambda DNA (1 g) is incubated with Taq DNA Polymerase in 50 l test buffer, overlaid with paraffin oil, at 65C for 16 h. The amount of enzyme that shows no degradation of the lambda DNA is stated under "Endo 1."
Absence of Endonucleases Eco RI/Hind III fragments (1 g) of lambda DNA is incubated with Taq DNA Polymerase in 50 l test buffer, overlaid with paraffin oil, at 65C for 16 h. The amount of enzyme that shows no alteration of the banding pattern is stated under "Endo 2." Absence of Nicking Activity Supercoiled pBR322 DNA (1 g) is incubated with Taq DNA Polymerase in 50 l test buffer, overlaid with paraffin oil, at 65C for 4 h. The amount of enzyme that shows no relaxation of the supercoiled DNA is stated under "Nick. Act." Absence of Exonucleases Different amounts of Taq DNA Polymerase are incubated in 100 l test buffer containing [3H]-labeled DNA, overlaid with paraffin oil, at 65C for 4 h. The amount of enzyme that shows no exonuclease activity is stated under "Exo."

242 910

25 ml (25 vials of 1 ml) 25 ml (1 vial of 25 ml)100 ml (4 vials of 25 ml) 843 001
Thin-walled PCR 1000 tubes (200 l) Tubes 1000 tubes (500 l) 050 001
NOTICE TO PURCHASER: LIMITED LICENSE A 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 use in research and development, has an up-front fee component and a runningroyalty component. The purchase price of the product includes a limited, nontransferable license under the running-royalty component to use only this amount of product to practice the Polymerase Chain Reaction (PCR) and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with 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 in order to have a complete license. These rights under the up-front fee component may be purchased from Applied Biosystems or obtained by purchasing an authorized thermal cycler. This product is also an authorized reagent and may also be used under service sublicenses from Applied Biosystems under the foregoing patents. No right to perform or offer commercial services of any kind using PCR, including without limitation reporting the results of purchasers activities for a fee or other commercial consideration, is hereby granted expressly, by implication or estoppel. This product is for research use only. Diagnostic uses require a separate license from Roche. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Supplementary Information

Conventions

Text Conventions To make information consistent and memorable, the following text conventions are used in this package insert: Text Convention Use Numbered Instructions Steps in a process that usually occur in the order listed labeled , ,etc. Numbered Instructions Steps in a procedure that must be performed in the labeled , ,etc. order listed Asterisk * Denotes a product available from Roche Applied Science
Symbols In this package insert the following symbols are used to highlight important information: Symbol L N 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.

Contact and Support

To ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site at:
www.roche-applied-science.com/support
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Trademarks FASTSTART and HIGH PURE are Trademarks of Roche.
Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim Germany

For general laboratory use. FOR IN VITRO USE ONLY.

Taq DNA Polymerase

From Thermus aquaticus BM, recombinant (E. coli) Deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7
Cat. No. Cat. No. Cat. No. Cat. No. Cat. No.

100 U 500 U U U U

Storage and stability Applications

Version 11, Jan. 2003

Store at 15 to 25 C

Product overview

Pack content Vial Content Taq DNA Poly- 20 l (100 U pack size) merase (5 U/l) 100 l (500 U pack size) l (4 x 250 U pack size) l (10 x 250 U pack size) l (20 x 250 U pack size) Enzyme storage buffer: 20 mM Tris-HCl, 1 mM dithiothreitol, 0.1 mM EDTA, 0.1 M KCl, 0.5% Nonidet P40 (v/v), 0.5% Tween20 (V/V), 50% glycerol (v/v), pH 8.0 (4C) PCR reaction 1 ml (100 U pack size) buffer, 1 ml (500 U pack size) conc., (with ml (4 x 250 U pack size) MgCl2) ml (10 x 250 U pack size) ml (20 x 250 U pack size) Buffer composition (10 x conc.): 100 mM Tris-HCl, 15 mM MgCl2, 500 mM KCl, pH 8.3 (20C) Product Description Taq DNA Polymerase (1-2) is a highly processive 5-3 DNA polymerase that lacks 3-5 exonuclease activity (3). It consists of a single polypeptide chain with a molecular weight of approx. 95 kD. Taq DNA Polymerase was originally isolated from the thermophilic eubacterium Thermus aquaticus BM, a strain lacking Taq I restriction endonuclease. The enzyme was cloned in E. coli and is isolated to be free of unspecific endo-or exonucleases.
Stable at 15 to 25C until the control date printed on the label. Polymerase Chain Reaction (PCR) DNA labeling reactions (5-6) Sequencing/ cycle sequencing (5,7)

Standard PCR procedure

General considerations The optimal conditions (incubation times and temperatures, concentrations of enzyme, template DNA, Mg2+) depend on the system used and have to be determined individually (8). In particular, the Mg2+ concentration and the amount of enzyme used per assay should be titrated for optimal efficiency of DNA synthesis. As a starting point, use the following guidelines: Optimal enzyme concentration: 0.5 2.5 U/50 l ; a concentration of 1.25 U will usually produce satisfactory results. Optimal Mg2+ concentration can vary from 1.5 mM to 5 mM; in most cases a Mg2+ concentration of 1.5 mM will produce satisfactory results (2-3) when using 200 M dNTP (each). dNTP concentration: always use balanced solutions of all four dNTP. The final concentration of each dNTP should be between 50 and 500 M; the most commonly used concentration is 200 M. Increase concentrations of Mg2+ when increasing the concentration of dNTP. Template concentration: e.g. human genomic DNA template: 10 ng-250 ng; plasmid DNA template: 0.1 ng-15 ng. The optimal buffer for the template DNA is either simply sterile double-distilled water or 5-10 mM Tris (pH 7-8). Avoid dissolving the template in TE buffer because EDTA chelates Mg2+. For a larger number of reactions, we recommend that you prepare two reaction mixes. This circumvents the need of hot start and avoids that the enzyme interacts with primers or template during the reaction set-up. It is also recommended to prepare a Master Mix for setting up multiple reactions. The Master Mix typically contains all of the components needed for all PCR tests to be performed at a volume 10% greater than that required for the total number of PCR assays.
Enzyme properties Volume activity Error rate* Optimal enzyme concentration Standard enzyme concentration Optimal pH Optimal elongation temperature Optimal Mg2+ concentration Standard Mg2+ concentration PCR product size 5 U/l approx. 1.310-5 varies from 0.5-2.5 U per 50 l reaction 1.25 U per 50 l reaction around 9 (adjusted at 20C) around 72C varies form 1.5 - 5 mM (as MgCl2) 1.5 mM (as MgCl2) when using 200 M dNTP each optimized for up to 3 kb. PCR possible up to 10 kb with yield diminishing as DNA fragment length increases TA-cloning; addition of A-overhangs accepts modified nucleotides like radiolabeled nucleotides, DIG-dUTP, biotin-dUTP yes Over 80% activity after 30 cycles (1 min 95C, 1 min 37C, 3 min 72C)

Preparation of reaction mixes
PCR Cloning Incorporation of modified nucleotides Prevention of carry-over contamination Thermostability
* Relative fidelity determined by the lacI assay (4).

0103.11180975

Step 1 2
Action Briefly centrifuge all reagents before starting. Prepare two mixes of reagents in sterile microfuge tubes (on ice): Mix 1 (for one reaction) Reagents Volume Final conc.
Thermal profile B: gradually increasing extension time; this procedure ensures a higher yield of amplification products. Temp. Time Cycle No. Initial denaturation Denaturation Annealing Elongation Denaturation Annealing Elongation 94Cb 2 min 1x 10 x
sterile add up to double-dist. 25 l water dNTP mix (10 mM) Upstream primer Downstream primer Template DNA Final vol. 1 l variable variable 200 M (of each dNTP) 0.1 0.6 M 0.1 0.6 M 0.nga
94C b( 15 s 30 s 55C-65Cc 30 s 60 s d 72C 45 s 3 min 94Cb 15 s 30 s 30 s 50C-65Cc d 72C 45 s 3 min + 5 s cycle elongation for each succ. cyclee 72Cd 4C 7 min

Final elongation Cooling

variable 25 l

unlimited time

Mix 2 (for one reaction) Reagents Volume Final conc. sterile double- 19.75 l distilled water PCR reaction 5 l 1x buffer, 10 x conc (1.5 mM MgCl2) Taq DNA Poly0.25 l 1.25 U/ merase, 5 U/l reaction Final volume 25 l 3 Combine Mix 1 and Mix 2 in a thinwalled PCR tube (on ice). Gently vortex the mixture to produce a homogeneous reaction, then centrifuge briefly to collect the sample at the bottom of the tube. Continue to thermal cycling immediately. Note: Carefully overlay the reaction with mineral oil if required by your type of thermal cycler.
The denaturation temperature can vary from 92C-95C. 94C is the standard denaturation temperature. c Optimal annealing temperature depends on the melting temperature of the primers and on the system used. d For PCR products up to 1 kb elongation temperature should be around 72C; for PCR products larger than 1 kb elongation temperature should be around 68C. e For example, cycle no. 11 is 5 s longer than cycle 10, cycle no. 12 is 10 s longer than cycle 10, cycle no. 13 is 15 s longer than cycle 10, etc.

2. After cycling, the samples may be frozen for later use. Possible further procedures: Check the PCR product on an agarose gel for size and specificity using an appropriate size marker. Purify the PCR product with the High Pure PCR Product Purification Kit (Cat. No. 676), e.g. before performing nested PCR.
E.g. human genomic DNA template: 10 ng-250 ng; plasmid DNA template: 0.1 ng-15 ng.

Thermal cycling

1. Place samples in the thermal cycler, and start cycling using the thermal profiles mentioned below. Thermal profile A: single extension time Initial denaturation Denaturation Annealing Elongation Final elongation Cooling Temp. 94Cb Time 2 min Cycle No. 1x x
94Cb 15 s 30 s 55C-65Cc 30 s 60 s d 72C 45 s 3 min 72Cd 7 min 4C

Roche Applied Science

Trouble shooting
Little or no PCR product Possible cause Difficult template e.g. GC-rich templates Recommendation Use the GC-RICH PCR System. Add DMSO (at 8%) and titrate enzyme concentration down to 0.5 U per reaction.

Unit assay

Unit definition One unit Taq DNA polymerase is defined as the amount of enzyme that incorporates 20 nmol of total deoxyribonucleoside-triphosphates into acid precipitable DNA within 60 min at 65C under the assay conditions stated below.
DNA template problems Check quality and concentration of template: Analyze an aliquot on an agarose gel to check for possible degradation. Make a control reaction on template with an established primer pair or PCR system. Check or repeat purification of template. Enzyme concentration too low Increase enzyme concentration to 2 U Taq DNA Polymerase per 50 l reaction. If necessary, increase the amount of polymerase in 0.5 U steps.
Unit assay: Incubation buffer for assay on activated DNA: assay on activated 67 mM Tris/HCl; pH 8.3/25C, 5 mM MgCl2, 10 mM DNA Mercaptoethanol, 0.2% Polydocanol, 0.2 mg/ml Gelatine, 0.2 mM each dATP, dGTP, dTTP and 0.1 mM dCTP, pH 8.3 (25C). Incubation procedure: M13mp9ss, M13 primer (17mer) and 1 Ci (-32P) dCTP are incubated with suitable dilutions of Taq DNA Polymerase in 50 l incubation buffer at 65C for 60 min. The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation.

MgCl2 concentration too Increase the MgCl2 concentration in steps of low 0.25 mM. (1.5 mM MgCl2 is the minimal concentration.) Cycle conditions not optimal Decrease annealing temperature. Increase cycle number. Make sure that the final elongation step was carried out. Design alternative primers.

Quality control

Each lot of Taq DNA polymerase is tested for contaminating activities as described in the following. Test buffer Absence of endonucleases 10 mM Tris-HCl, 1,5 mM MgCl2, 50 mM KCl, pH 8.3 (20C). 1 g lambda DNA is incubated with Taq DNA polymerase in 50 l test buffer at 65C for 16 h. The amount of enzyme showing no degradation of the lambda DNA is stated under "Endo 1". 1 g Eco RI/Hind III-fragments of lambda DNA is incubated with Taq DNA polymerase in 50 l test buffer at 65C for 16 h. The amount of enzyme showing no alteration of the banding pattern is stated under "Endo 2". 1 g supercoiled pBR322 DNA is incubated with Taq DNA polymerase in 50 l test buffer at 65C for 16 h. The amount of enzyme showing no alteration of the no relaxation of supercoiled DNA is stated under "Nick.Act.". Different amounts of Taq DNA polymerase are incubated in 100 l test buffer containing (3H)-labeled DNA at 65C for 4 h. The amount of enzyme showing no exonuclease-activity is stated under "Exo".
Primer design not optimal
Primer concentration not Both primers must have the same concentraoptimal tion. Titrate primer concentration (0.1 0.6 M). Primer quality or storage If you use an established primer pair, check problems performance on an established PCR system (control template). Make sure that the primers are not degraded. Always store primers at 15 to 25C. Formation of primer dimers Use FastStart Taq DNA Polymerase. Use two reaction mixes according to the protocol above.

Absence of endonucleases

Absence of "nicking activity"
Multiple bands or background smear Possible cause Annealing temperature too low Primer design or concentration not optimal Recommendation Increase annealing temperature according to the primer length. Review primer design. Titrate primer concentration (0.1 0.6 M). Both primers must have the same concentration. Perform nested PCR with nested primers. Perform PCR with GC-RICH solution. Absence of exonucleases

References

Difficult template e.g. GC- rich template

DNA template problems Use serial dilution of template. PCR products in negative control experiments Possible cause Carry-over contamination Recommendation Exchange all reagents, especially water. Use aerosol-resistant pipette tips. Set up PCR reactions in an area separate from that used for PCR product analysis. Use dUTP (600 M) instead of dTTP (200 M) in combination with thermolabile UNG (1 U/ 50 l reaction) and increase Mg2+ concentration up to 4 mM at most.
1 Chien, A., Edgar, D.B. and Trela, J.M. (1976) J. Bacteriol. 127, 1550-1557. 2 Lawyer, F.C. et al. (1989) J. Biol. Chem. 264, 6427-6437. 3 Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 60086013. 4 Frey, B. and Suppmann, B. (1995) Biochemica 2, 8-9. 5 Innis, M.A. et al. (1988) Proc. Natl. Acad. Sci USA 85, 94369440. 6 Lo, Y.-M.D., Mehal, W.Z. and Fleming, K.A. (1988) Nucleic Acids Res. 16, 8719. 7 Taq Polymerase: increased enzyme versatility in DNA sequencing (1988) Applied Biosystems. 8 Erlich, H.A. (ed.) (1989) PCR Technology: Principles and Application for DNA Amplification, Stockton Press, New York.
Specific problems in RT-PCR application Possible cause No product, additional bands, background smear Recommendation The volume of cDNA template (RT-reaction) should not exceed 10% of the final volume of the PCR reaction. Follow trouble shooting above. Increase MgCl2 by titration in steps of 0.25 mM.

Related Products

Roche Applied Science offers a large selection of enzymes, reagents, and systems for PCR and RT-PCR assays. For a complete overview of our products and for more detailed information on PCR and RT-PCR please visit and bookmark our Amplification Special Interest Site at http://www.roche-applied-science.com/PCR. PCR Product Selection Guide: Application Standard PCR Product Size [kb] 3 Recommended Product Taq DNA Polymerase Pack size 100 U 500 U 4x250 U 10x250 U 20x250 U 100 U 2x250 U 10x250 U 50 U 100 U 500 U 4x250 U 10x250 U 20x250 U 50 react. 100 react. 250 react. 100 U 2x250 U 10x250 U 100 U 2x250 U 10x250 U 100 U 100 U Cat. No. 306

Notice to purchaser

A license under U.S. Patents 4,683,202, 4,683,195, and 4,965,188 or their foreign counterparts, owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd ("Roche"), has an up-front fee component and a running-royalty component. The purchase price of this product includes limited, nontransferable rights under the running-royalty component to use only this amount of the product to practice the Polymerase Chain Reaction ("PCR") and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the upfront fee component. Rights to the up-front fee component must be obtained by the end user in order to have a complete license to use this product in the PCR process. These rights under the up-front fee component may be purchased from Applied Biosystems or obtained by purchasing an Authorized Thermal Cycler. No right to perform or offer commercial services of any kind using PCR, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is hereby granted by implication or estoppel. Further information on purchasing licenses to practice the PCR Process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404 or the Licensing Department, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501.

High Specificity PCR

Expand High Fidelity PCR System FastStart Taq DNA Polymerase (for hot start PCR)
How to contact Roche Applied Science
www.roche-applied-science.com to order, solve technical queries, find product information, or contact your local sales representative. www.roche-applied-science.com/pack-insert/1146165a.pdf

High Fidelity PCR

Tgo DNA Polymerase
Expand High Fidelity PCR System Expand Long Template PCR System Expand 20 kb PLUS PCR System GC RICH PCR System

Long Template PCR

Difficult DNA Template PCR
Expand Long Template PCR System
100 U 834 2x250 U 842 10x250 U 060
PCR Nucleotide Selection: Product Description Pack size Cat. No.
Set of Deoxy-Nucleotides, Separate vials of dATP, 4 x PCR Grade dCTP, dGTP, and dTTP. mol (mM each l) PCR Nucleotide Mix Premixed ready-to-use 200 l 295 solution of PCR Grade 10 x 200 l 362 dATP, dCTP, dGTP, and dTTP. 10 mM. Premixed ready-to-use 2 x 100 l solution of PCR Grade dATP, dGTP, dTTP (each 10 mM), and dUTP (30 mM) 814 362

PCR Nucleotide MixPLUS

Expand, FastStart, and GeneAmp are trademarks of a member of the Roche Group. Tween 20 is a trademark of ICI Americas Inc., Wilmington, USA. Nonidet P40 is a trademark of Shell International Petroleum Company Limited, U.K.
Roche Diagnostics GmbH Roche Applied Science Nonnenwald Penzberg Germany