For general laboratory use.
FOR IN VITRO USE ONLY.
Taq DNA Polymerase, dNTPack
5 U/ l Taq DNA Polymerase with ready-to-use PCR Grade Nucleotide Mix
Cat. No. 04 728 866 001 100 U
Cat. No. 04 728 874 001 500 U
Cat. No. 04 728 882 001 4 × 250 U
Cat. No. 04 728 904 001 10 × 250 U
Cat. No. 04 728 858 001 20 × 250 U Version July 2005
Store the kit at 15 to 25°C
1. What this Product Does Application
• Polymerase Chain Reaction (PCR): Taq DNA Polymerase activity is
Number of Reactions stable during prolonged incubation at high temperatures (95°C)
If 1.25 U are used per 50 l reaction, Taq DNA Polymerase, dNTPack is and can therefore be used to amplify DNA fragments by PCR.
designed for: • DNA labeling reactions (4, 5)
• approx. 80 reactions (Cat. No. 04 728 866 001) • Sequencing / cycle sequencing (4, 6)
• approx. 400 reactions (Cat. No. 04 728 874 001)
• approx. 800 reactions (Cat. No. 04 728 882 001) Enzyme Properties
• approx. 2000 reactions (Cat. No. 04 728 904 001) Volume Activity 5 U/ l
• approx. 4000 reactions (Cat. No. 04 728 858 001) Optimal Enzyme Concentra- Varies between 0.5 and 2.5 U per 50 l
Contents Standard Enzyme Concentra- 1.25 U per 50 l reaction
Label Contents tion
Taq DNA Poly- • 20 l (100 U pack size) Optimal pH Around 9 (adjusted at 20°C)
merase • 100 l (500 U pack size) Optimal Elongation Tempera- Around 72°C
(5 U/ l) • 4 × 50 l (4 x 250 U pack size) ture
• 10 × 50 l (10 x 250 U pack size) Optimal Mg2+ Concentration Varies between 1.5 and 5 mM (as
• 20 × 50 l (20 x 250 U pack size) MgCl2)
Enzyme storage buffer: 20 mM Tris-HCl, 1 mM Standard Mg2+ Concentration 1.5 mM (as MgCl2) when used with
dithiothreitol, 0.1 mM EDTA, 0.1 M KCl, 0.5% Noni- 200 M of each dNTP
det P40 (v/v), 0.5% Tween 20 (v/v), 50% glycerol
(v/v), pH 8.0 (4°C) Size of PCR Products Enzyme optimally amplifies up to 3 kb
products. (PCR is possible up to 10 kb,
PCR reaction • 1 ml (100 U pack size) but yield diminishes as DNA fragment
buffer with • 3 × 1 ml (500 U pack size) length increases.)
MgCl2 ,10 × • 6 × 1 ml (4 x 250 U pack size)
conc. PCR Cloning T/A-cloning (Enzyme adds a single,
• 15 × 1 ml (10 x 250 U pack size) overhanging A.)
• 30 × 1 ml (20 x 250 U pack size)
Buffer composition: 100 mM Tris-HCl, 15 mM Incorporation of Modified Enzyme accepts modified nucleotides
MgCl2, 500 mM KCl, pH 8.3 (20°C) Nucleotides like radiolabeled nucleotides, DIG-
PCR Grade • 1 × 200 l (100 U pack size)
Nucleotide Mix • 4 × 200 l (500 U pack size) Thermostability Enzyme retains over 80% activity after
30 cycles (1 min 95°C, 1 min 37°C,
• 8 × 200 l (4 x 250 U pack size) 3 min 72°C).
• 20 × 200 l (10 x 250 U pack size)
• 40 × 200 l (20 x 250 U pack size)
Ready-to-use 10 mM deoxynucleotide solution 2. How To Use this Product
Storage and Stability 2.1 Before You Begin
The undiluted solutions are stable when stored at –15 to –25°C General considerations
through the control date printed on the label.
The optimal conditions (incubation times and temperatures, concen-
Additional Equipment and Reagents Required tration of enzyme, template DNA, Mg2+) vary from system to system
and must be determined for each individual experimental system (7).
• Template DNA, gene-specific PCR primer pair At the very least, you should titrate the Mg2+ concentration and the
• Water, PCR Grade* amount of enzyme used per assay to ensure optimal efficiency of DNA
• Thermal block cycler (e.g., Applied Biosystems GeneAmp PCR Sys- synthesis.
• 0.2 ml thin-walled PCR tubes*
• Sterile reaction tubes for preparing master mixes and dilutions * available from Roche Applied Science
Roche Applied Science
As a starting point, use the following guidelines: 2.3 PCR
• Optimal enzyme concentration: 0.5 – 2.5 U/50 l. A concentration of
1.25 U/50 l will usually produce satisfactory results. ³ • For each reaction, combine 25 l Master Mix 1 and 25 l Mas-
ter Mix 2 in a thin-walled PCR tube on ice.
• Optimal Mg2+ concentration can vary between 1.5 mM and 5 mM. • Gently vortex the mixture to produce a homogeneous reaction,
In most cases a Mg2+ concentration of 1.5 mM will produce satis- then centrifuge briefly to collect the solution at the bottom of
factory results (2, 3) if you use 200 M of each dNTP. the tube.
• dNTP concentration: Always use equal concentrations of all four
N Start thermal cycling immediately. Do not store complete
dNTPs. The final concentration of each dNTP should be between 50 reaction mixes on ice.
and 500 M; the most commonly used concentration is 200 M. If
you increase the dNTP concentration, you must also increase the · Place your samples in a thermal block cycler and use either of
Mg2+ concentration. the thermal profiles below to perform PCR.
• Template concentration: Typical concentrations are 10 ng - 250 ng • Thermal Profile A: fixed extension time
human genomic DNA and 0.1 ng - 15 ng plasmid DNA. Cycles Time Temp
• The optimal buffer for the template DNA is either sterile double-dis-
Initial Denaturation 1× 2 min 94°C
tilled water or 5 - 10 mM Tris (pH 7 - 8).
N Do not dissolve the template in TE buffer because EDTA che-
Denaturation 25 – 30× 15 s - 30 s 94°C
Annealing 30 s - 60 s 55 to 65°C
lates Mg2+. Elongation 45 s – 3 min 72 or 68°C
2.2 Preparation of Reaction Mixes Final Elongation 1× 7 min 72 or 68°C
For multiple reactions, we recommend that you prepare two reaction Cooling indefinitely 4°C
mixes. This eliminates the need for a hot start and keeps the enzyme
from interacting with primers and template during preparation of the • Thermal Profile B: gradually increasing extension time (This
reaction mixes. If you are setting up multiple reactions, we also recom- procedure ensures a higher yield of amplification products.)
mend preparing a Master Mix that contains all reaction components Cycles Time Temp
that are present in each reaction. The volume of each Master Mix typi-
cally should be 110% of the volume needed for all the samples. (For Initial Denaturation 1× 2 min 94°C
example to prepare Master Mix 2 below for 10 reactions, make 275 l Denaturation 10 × 15 s - 30 s 94°C
of the mix.) (The extra volume allows for losses during pipetting.) Annealing 30 s - 60 s 55 to 65°C
Elongation 45s – 3 min 72 or 68°C
Preparation of Master Mix 1
Denaturation 15 – 20× 15 s - 30 s 94°C
³ • Thaw the reagents and store on ice. Annealing 30 s 55 to 65°C
• Briefly vortex and centrifuge all reagents before setting up the Elongation 45 s – 3 min + 5 s 72 or 68°C
reactions. cycle elongation for
each succ. cyclea
Prepare a 10× conc. solution of the PCR primers.
Final Elongation 1× 7 min 72 or 68°C
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 concen- Cooling indefinitely 4°C
tration of each primer. a Forexample, cycle no. 11 is 5 s longer than cycle 10, cycle no. 12 is 10 s
To a sterile reaction tube on ice, add the components in the longer than cycle 10, cycle no. 13 is 15 s longer than cycle 10, etc.
order listed below: (For each 50 l reaction) L The denaturation temperature can vary between 92°C and 95°C. The
standard denaturation temperature is 94°C.
Component Volume Final conc. Optimal annealing temperature depends on the melting temperature of
the primers and on the experimental system.
PCR Grade Nucleotide Mix 1 l 200 M (of each For PCR products up to 1 kb, elongation temperature should be around
dNTP) 72°C; for PCR products larger than 1 kb, elongation temperature should
be around 68°C.
PCR primer mix, 10× 5 l 0.1 – 0.6 M
Template DNA variable 100 – 250 ng After cycling, if the samples are not used immediately, store
them frozen for later use.
Water, PCR Grade to make a final L For best results, do the following:
vol. of 25 l
• Check the PCR product on an agarose gel for size and specific-
Final volume 25 l ity. Use an appropriate size marker.
Mix and centrifuge briefly. • Purify the PCR product with the High Pure PCR Product Purifi-
cation Kit* (e.g., before performing nested PCR).
Preparation of Master Mix 2
³ • Thaw the reagents and store on ice.
• Briefly vortex and centrifuge all reagents before setting up the
· To a sterile reaction tube on ice, add the components in the
order listed below: (For each 50 l reaction)
Component Volume Final conc.
PCR reaction buffer, 10× 5 l 1×
(1.5 mM MgCl2)
Taq DNA Polymerase (5 U/ l) 0.25 l 1.25 U/reaction
Water, PCR Grade 19.75 l
Final volume 25 l
» Mix and centrifuge briefly.
Roche Applied Science
Possible Cause Recommendation
Possible Cause Recommendation PCR pro- Carryover contami- • Replace all reagents, espe-
ducts in ne- nation cially water.
Little or no Difficult template • Perform PCR with GC-RICH gative control • Use aerosol-resistant pipette
PCR product e.g., GC-rich tem- PCR System*. experiments tips.
plates • Add DMSO (final concentra- • Set up PCR reactions in an area
tion, 8%) and reduce enzyme separate from that used for
concentration (e.g. use as little PCR product analysis.
as 0.5 U per reaction). • To eliminate carryover contam-
DNA template pro- Check quality and concentration inants: Use dUTP* (600 M)
blems of template: instead of dTTP (200 M) and
• Analyze an aliquot on an agar- thermolabile UNG* (1 U/50 l
ose gel to check for possible reaction); also, increase Mg2+
degradation. concentration (to a maximum
• Test the template with an of 4 mM) to compensate for
established primer pair or PCR higher dNTP conc.
system. Problems No product, addi- • The volume of cDNA template
• Check or repeat template puri- specific to tional bands, back- (RT-reaction) should not
fication. RT-PCR ground smear exceed 10% of the final volume
Enzyme concentra- • Increase enzyme concentra- of the PCR reaction.
tion too low tion to 2 U Taq DNA Poly- • Follow troubleshooting tips
merase per 50 l reaction. above.
• If necessary, increase the • Increase MgCl2 in 0.25 mM
amount of polymerase in 0.5 U steps.
MgCl2 concentra- Increase the MgCl2 concentra- 4. Additional Information on this Product
tion too low tion in 0.25 mM steps. (The mini-
mal acceptable concentration is Product Description
1.5 mM MgCl2.)
Taq DNA Polymerase (1, 2) is a highly processive 5’→3’ DNA poly-
Cycle conditions not • Decrease annealing tempera- merase that lacks 3’→5’ exonuclease activity (3). It is a single polypep-
optimal ture. tide chain with a molecular weight of approx. 95 kD.
• Increase cycle number. Taq DNA Polymerase was originally isolated from the thermophilic
• Make sure that the final elon- eubacterium Thermus aquaticus BM, a strain lacking Taq I restriction
gation step is included in the endonuclease. The enzyme preparation obtained from E. coli is free of
program. nonspecific endo- or exonucleases.
Primer design not Design alternative primers. Unit Definition
One unit Taq DNA polymerase is defined as the amount of enzyme
Primer concentra- • Both primers must have the that incorporates 10 nmol of total deoxyribonucleosidetriphosphates
tion not optimal same concentration. into acid precipitable DNA within 30 min at 75°C under the assay con-
• Titrate primer concentration ditions givenabove.
(0.1 – 0.6 M).
Primer quality or • If you use an established Unit Assay
storage problems primer pair, check performance Incubation buffer: 67 mM Tris/HCl; pH 8.3/25°C, 5 mM MgCl2, 10
in an established PCR system mM Mercaptoethanol, 0.2% Polydocanol, 0.2 mg/ml Gelatine, 0.2 mM
(e.g. with a control template). each dATP, dGTP, dTTP and 0.1 mM dCTP.
• Make sure that the primers are Incubation procedure: M13mp9ss, M13 primer (17mer) and 1 Ci
not degraded. (α-32P) dCTP are incubated with suitable dilutions of Taq DNA poly-
• Always store primers at –15 to merase in 50 l incubation buffer at 65°C for 60 min. The amount of
–25°C. incorporated dNTPs is determined by trichloroacetic acid precipitation.
Formation of primer • Use two Master Mixes, as 4.1 References
dimers directed in the protocol above. 1 Chien, A., Edgar, D. B. & Trela, J. M. (1976) Deoxyribonucleic acid
• Use FastStart Taq DNA Poly- polymerase from the extreme thermophile Thermus aquaticus. J.
merase* instead of Taq DNA Bacteriol. 127, 1550-1557.
Polymerase. 2 Lawyer, F. C. et al. (1989) Isolation, characterization and expres-
Multiple Annealing tempera- Increase annealing temperature sion in Escherichia coli of the DNA polymerase gene from the
bands or ture too low (Longer primers have higher extreme thermophile Thermus aquaticus. J. Biol. Chem. 264,
background annealing temperatures). 6427-6437.
smear Primer design or • Review primer design. 3 Tindall, K. R. & Kunkel, T. A. (1988) Fidelity of DNA synthesis by
concentration not the Thermus aquaticus DNA polymerase. Biochemistry 27, 6008-
• Titrate primer concentration
(0.1 – 0.6 M).
4 Innis, M. A., et al. (1988) DNA sequencing with Thermus aquat-
• Both primers must have the
icus DNA polymerase and direct sequencing of polymerase
chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85,
• Perform nested PCR with 9436-9440.
5 Lo, Y.-M. D., Mehal, W. Z. & Fleming, K. A. (1988) Rapid produc-
Difficult template Perform PCR with GC-RICH PCR tion of vector-free biotinylated probes using the polymerase
(e.g., GC- rich tem- System*. chain reaction. Nucleic Acids Res. 16, 8719.
plate) 6 Taq polymerase: increased enzyme versatility in DNA
DNA template Use serial dilution of template. sequencing (1988) Applied Biosystems.
Roche Applied Science
7 Erlich, H. A. (ed.) (1989) PCR Technology: Principles and Appli- 5.2 Ordering Information
cation for DNA Amplification, Stockton Press, New York. Roche Applied Science offers a large selection of reagents and sys-
8 Mesquita, P. (2003) Human MUC2 mucin gene is transcription- tems for life science research. For a complete overview of related
ally regulated by cdx homeodomain proteins in gastrointestinal products and manuals, please visit and bookmark our home page
carcinoma cell lines. J. Biol. Chem. 278: 51549-51556. www.roche-applied-science.com and our Special Interest Sites includ-
9 Zhu, Y. (2002) Hemin induces neuroglobin expression in neural ing: http://www.roche-applied-science.com/PCR/
cells. Blood 100: 2494-2498.
Product Pack Size Cat. No.
4.2 Quality Control DNA High Pure PCR 100 purifications 11 796 828 001
Each lot of Taq DNA Polymerase is tested for contaminating Purifica- Template Purifica-
activities as described in the following: tion tion Kit
High Pure PCR 50 purifications 11 732 668 001
Test Buffer Product Purifica- 250 purifications 11 732 676 001
10 mM Tris-HCl, 1.5 mM MgCl2 , 50 mM KCl, pH 8.3 (20°C). tion Kit
Addi- Digoxigenin-11- 25 nmol (25 l) 11 573 152 910
Absence of Endonucleases tional dUTP 125 nmol (125 l) 11 573 179 910
Lambda DNA (1 g) is incubated with Taq DNA Polymerase in
50 l test buffer, overlaid with paraffin oil, at 65°C for 16 h. The Digoxigenin-11- 25 nmol (25 l) 11 093 088 910
amount of enzyme that shows no degradation of the lambda dUTP
DNA is stated under "Endo 1." (alkali-stable)
Biotin-16-dUTP 50 nmol (50 l) 11 093 070 910
Absence of Endonucleases Fluorescein-12- 25 nmol (25 l) 11 373 242 910
Eco RI/Hind III fragments (1 g) of lambda DNA is incubated with dUTP
Taq DNA Polymerase in 50 l test buffer, overlaid with paraffin oil, at
65°C for 16 h. The amount of enzyme that shows no alteration of the Water, 25 ml 03 315 932 001
banding pattern is stated under "Endo 2." PCR Grade (25 vials of 1 ml)
25 ml (1 vial of 25 ml)03 315 959 001
Absence of ”Nicking Activity” 100 ml
(4 vials of 25 ml) 03 315 843 001
Supercoiled pBR322 DNA (1 g) is incubated with Taq DNA Poly-
merase in 50 l test buffer, overlaid with paraffin oil, at 65°C for 4 h. Thin-walled PCR 1000 tubes (200 l) 11 667 041 001
The amount of enzyme that shows no relaxation of the supercoiled Tubes 1000 tubes (500 l) 11 667 050 001
DNA is stated under "Nick. Act."
Disclaimer of License: Notice to Purchaser
Absence of Exonucleases 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
Different amounts of Taq DNA Polymerase are incubated in 100 l test in research and development, has an up-front fee component and a running-
buffer containing [3H]-labeled DNA, overlaid with paraffin oil, at 65°C royalty component. The purchase price of the product includes limited, non-
for 4 h. The amount of enzyme that shows no exonuclease activity is transferable rights under the running-royalty component to use only this
stated under "Exo." amount of product to practice the Polymerase Chain Reaction (PCR) and
related processes described in said patents solely for the research and devel-
opment activities of the purchaser when this product is used in conjunction
5. Supplementary Information 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 compo-
5.1 Conventions nent may be purchased from Applied Biosystems or obtained by purchasing an
authorized thermal cycler. No right to perform or offer commercial services of
Text Conventions any kind using PCR, including without limitation reporting the results of pur-
To make information consistent and memorable, the following text conventions chaser's activities for a fee or other commercial consideration, is hereby
are used in this package insert: granted by implication or estoppel. Further information on purchasing licenses
to practice the PCR process may be obtained by contacting the Director of
Text Convention Use Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California
Numbered Instructions Steps in a process that usually occur in the order listed 94404, USA.
labeled , ,etc.
Numbered Instructions Steps in a procedure that must be performed in the
labeled ³, ·,etc. order listed HIGH PURE is a trademark of Roche.
Asterisk * Denotes a product available from Roche Applied
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