Taq DNA Polymerase (recombinant)

Catalog Number EP0401, EP0402, EP0404, EP0405, EP0406

Pub. No. MAN0012031 Rev. B.00

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Contents and storage

Cat. No. Contents Amount Storage
Taq DNA Polymerase, 5 U/µL 100 U
10X Taq Buffer with KCl 0.6 mL
EP0401
10X Taq Buffer with (NH4)2SO4 0.6 mL
25 mM MgCl2 0.6 mL
Taq DNA Polymerase, 5 U/µL 500 U
10X Taq Buffer with KCl 2 x 1.25 mL
EP0402
10X Taq Buffer with (NH4)2SO4 2 x 1.25 mL
25 mM MgCl2 2 x 1.25 mL
Taq DNA Polymerase, 1 U/µL, LC 500 U
10X Taq Buffer with KCl 2 x 1.25 mL
EP0404 -25 °C to -15 °C
10X Taq Buffer with (NH4)2SO4 2 x 1.25 mL
25 mM MgCl2 2 x 1.25 mL
Taq DNA Polymerase, 5 U/µL 5 x 500 U
10X Taq Buffer with KCl 10 x 1.25 mL
EP0405
10X Taq Buffer with (NH4)2SO4 10 x 1.25 mL
25 mM MgCl2 10 x 1.25 mL
Taq DNA Polymerase, 5 U/µL 10 x 500 U
10X Taq Buffer with KCl 20 x 1.25 mL
EP0406
10X Taq Buffer with (NH4)2SO4 20 x 1.25 mL
25 mM MgCl2 20 x 1.25 mL
Description
Taq DNA Polymerase is a highly thermostable DNA polymerase of the thermophilic bacterium Thermus aquaticus. The
enzyme catalyzes 5’→3’ synthesis of DNA, has no detectable 3’→5’ exonuclease (proofreading) activity and possesses
5’→3’ exonuclease activity. In addition, Taq DNA Polymerase exhibits deoxynucleotidyl transferase activity, which frequently
results in the addition of extra adenines at the 3’-end of PCR products.
Recombinant Taq DNA Polymerase is ideal for standard PCR of amplicons 5 kb or shorter.
Applications
• Routine PCR amplification of DNA fragments up to 5 kb (1).
• Generation of PCR product for TA cloning.
• DNA labeling (2-4).
• DNA sequencing (5).
Source
E.coli cells with a cloned pol gene from Thermus aquaticus YT1.

For Research Use Only. Not for use in diagnostic procedures.

Definition of Activity Unit
One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction in 30 min at 74°C.
10X Taq Buffer with KCl
100 mM Tris-HCl (pH 8.8 at 25°C), 500 mM KCl, detergent.
10X Taq Buffer with (NH4)2SO4
750 mM Tris-HCl (pH 8.8 at 25°C), 200 mM (NH4)2SO4, detergent.
Inhibition and Inactivation
• Inhibitors: ionic detergents (deoxycholate, sarkosyl and SDS) at concentrations higher than 0.06, 0.02 and 0.01 %, respectively (6).
• Inactivated by phenol/chloroform extraction.
Protocol
To prepare several parallel reactions and to minimize the possibility of pipetting errors, prepare a PCR master mix by mixing water,
buffer, dNTPs, primers and Taq DNA Polymerase. Prepare sufficient master mix for the number of reactions plus one extra. Aliquot the
master mix into individual PCR tubes and then add template DNA.
1. Gently vortex and briefly centrifuge all solutions after thawing.
2. Place a thin-walled PCR tube on ice and add the following components for each 50 µL reaction:
Contents Volume
10X Taq Buffer 5 µL
dNTP Mix, 2 mM each (#R0241) 5 µL (0.2 mM of each)
Forward primer 0.1-1.0 µM
Reverse primer 0.1-1.0 µM
25 mM MgCl2* 1-4 mM
Template DNA 10 pg - 1 µg
Taq DNA Polymerase 1.25 U
Water, nuclease-free (#R0581) to 50 µL
Total volume 50 µL
*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:
Final concentration of MgCl2, mM 1 1.5 2 2.5 3 4
Volume of 25 mM MgCl2 for 50 µL reaction, µL 2 3 4 5 6 8

3. Gently vortex and spin down the samples.

4. When using a thermal cycler that does not use a heated lid, overlay the reaction mixture with 25 µL of mineral oil.
5. Perform PCR using recommended thermal cycling conditions:
Step Temperature, °C Time Number of cycles
Initial denaturation 95 1-3 min 1
Denaturation 95 30 s
Annealing Tm-5 30 s 25-40
Extension 72 1 min/kb
Final Extension 72 5-15 min 1

Guidelines for Preventing Contamination of PCR Reaction

During PCR more than 10 million copies of template DNA are generated. Therefore, care must be taken to avoid contamination with other
templates and amplicons that may be present in the laboratory environment. General recommendations to lower the risk of contamination
are as follows:
• Prepare your DNA sample, set up the PCR mixture, perform thermal cycling and analyze PCR products in separate areas.
• Set up PCR mixtures in a laminar flow cabinet equipped with an UV lamp.
• Wear fresh gloves for DNA purification and reaction set up.
• Use reagent containers dedicated for PCR. Use positive displacement pipettes or use pipette tips with aerosol filters to prepare DNA
samples and perform PCR set up.
• Use PCR-certified reagents, including high quality water (e.g., Water, nuclease-free, (#R0581)).
• Always perform “no template control” (NTC) reactions to check for contamination.

2 Taq DNA Polymerase (recombinant)

Guidelines for Primer Design
Use primer design software or follow general recommendations for PCR primer design as outlined below:
• PCR primers are generally 15-30 nucleotides long.
• Optimal GC content of the primer is 40-60 %. Ideally, C and G nucleotides should be distributed uniformly along the primer.
• Avoid placing more than three G or C nucleotides at the 3’-end to lower the risk of non-specific priming.
• If possible, the primer should terminate with a G or C at the 3’-end.
• Avoid self-complementary primer regions, complementarities between the primers and direct primer repeats to prevent hairpin formation
and primer dimerization.
• Check for possible sites of undesired complementary between primers and template DNA.
• When designing degenerate primers, place at least 3 conservated nucleotides at the 3’-end.
• Differences in melting temperatures (Tm) between the two primers should not exceed 5 °C.
Estimation of Primer Melting Temperature
For primers containing less than 25 nucleotides, the approx. melting temperature (Tm) can be calculated using the following equation:
Tm= 4 (G + C) + 2 (A + T),
where G, C, A, T represent the number of respective nucleotides in the primer.
If the primer contains more than 25 nucleotides specialized computer programs are recommended to account for interactions of adjacent
bases, effect of salt concentration, etc.
Components of the Reaction Mixture
Template DNA
Optimal amounts of template DNA in the 50 µL reaction volume are 0.01-1 ng for both plasmid and phage DNA, and 0.1-1 µg for genomic
DNA. Higher amount of template increases the risk of generation of non-specific PCR products. Lower amount of template reduces the
accuracy of the amplification.
All routine DNA purification methods are suitable for template preparation. Trace amounts of certain agents used for DNA purification, such
as phenol, EDTA and proteinase K, can inhibit DNA polymerases. Ethanol precipitation and repeated washes of the DNA pellet with 70 %
ethanol normally removes trace contaminants from DNA samples.
MgCl2 Concentration
Due to the binding of Mg2+ to dNTPs, primers and DNA templates, Mg2+ concentration needs to be optimized for maximal PCR yield. The
recommended concentration range is 1-4 mM. If the Mg2+ concentration is too low, the yield of PCR product could be reduced. On the
contrary, non-specific PCR products may appear and the PCR fidelity may be reduced if the Mg2+ concentration is too high.
Taq DNA Polymerase is supplied with two buffers: Taq buffer with KCl and Taq buffer with (NH4)2SO4. K+ stabilizes primer annealing
whereas NH4+ has a destabilizing effect especially on weak hydrogen bonds between mismatched primer-template base pairs. Therefore for
standard PCR with Taq DNA Polymerase and 0.2 mM dNTPs the recommended MgCl2 concentrations are in general lower 1.5±0.25 mM
when using Taq buffer with KCl compared to 2.0±0.5 mM when using Taq buffer with (NH4)2SO4. Due to antagonistic effects of NH4+ and
Mg2+, Taq buffer with (NH4)2SO4 offers higher primer specificity in a broad range of magnesium concentrations at variety of annealing
temperatures.
If the DNA samples contain EDTA or other metal chelators, the Mg2+ ion concentration in the PCR mixture should be increased accordingly
(1 molecule of EDTA binds one Mg2+).
dNTPs
The recommended final concentration of each dNTP is 0.2 mM. In certain PCR applications, higher dNTP concentrations may be necessary.
Due to the binding of Mg2+ to dNTPs, the MgCl2 concentration needs to be adjusted accordingly. It is essential to have equal concentrations
of all four nucleotides (dATP, dCTP, dGTP and dTTP) present in the reaction mixture.
To achieve 0.2 mM concentration of each dNTP in the PCR mixture, use the following volumes of dNTP mixes:
dNTP Mix, dNTP Mix, dNTP Mix,
Volume of PCR mixture
2 mM each (#R0241) 10 mM each (#R0191) 25 mM each (#R1121)
50 µL 5 µL 1 µL 0.4 µL
25 µL 2.5 µL 0.5 µL 0.2 µL
20 µL 2 µL 0.4 µL 0.16 µL
Primers
The recommended concentration range of the PCR primers is 0.1-1 µM. Excessive primer concentrations increase the probability of
mispriming and generation of non-specific PCR products.
For degenerate primers higher primer concentrations in the range of 0.3-1 µM are often favorable.

3 Taq DNA Polymerase (recombinant)

Cycling Parameters
Initial DNA Denaturation
It is essential to completely denature the template DNA at the beginning of PCR to ensure efficient utilization of the template during the first
amplification cycle. If the GC content of the template is 50 % or less, an initial 1-3 min denaturation at 95 °C is sufficient. For GC-rich
templates this step should be prolonged up to 10 min. If longer initial denaturation step is required, or the DNA is denatured at a higher
temperature, Taq DNA Polymerase should be added after the initial denaturation step to avoid a decrease in its activity.
Denaturation
A DNA denaturation time of 30 seconds per cycle at 95 °C is normally sufficient. For GC-rich DNA templates, this step can be prolonged to
3-4 min. DNA denaturation can also be enhanced by the addition of either 10-15 % glycerol or 10 % DMSO, 5 % formamide or 1-1.5 M
betaine. The melting temperature of the primer-template complex decreases significantly in the presence of these reagents. Therefore, the
annealing temperature has to be adjusted accordingly.
In addition, 10 % DMSO and 5 % formamide inhibit DNA polymerases by 50 %. Thus, the amount of the enzyme should be increased if
these additives are used.
Primer Annealing
The annealing temperature should be 5 °C lower than the melting temperature (Tm) of the primers. Annealing for 30 seconds is normally
sufficient. If non-specific PCR products appear, the annealing temperature should be optimized stepwise in 1-2 °C increments. When
additives which change the melting temperature of the primer-template complex are used (glycerol, DMSO, formamide and betaine), the
annealing temperature must also be adjusted.
Extension
The optimal extension temperature for Taq DNA Polymerase is 70-75 °C. The recommended extension step is 1 min at 72 °C for PCR
products up to 2 kb. For larger products, the extension time should be prolonged by 1 min/kb.
Number of Cycles
The number of cycles may vary depending on the amount of template DNA in the PCR mixture and the expected PCR product yield.
If less than 10 copies of the template are present in the reaction, about 40 cycles are required. For higher template amounts, 25-35 cycles
are sufficient.
Final Extension
After the last cycle, it is recommended to incubate the PCR mixture at 72 °C for additional 5-15 min to fill-in any possible incomplete reaction
products. If the PCR product will be cloned into TA vectors, the final extension step may be prolonged to 30 min to ensure the highest
efficiency of 3’-dA tailing of PCR product. If the PCR product will be used for cloning using Thermo Scientific CloneJET PCR Cloning Kit
(#K1231), the final extension step can be omitted.
Troubleshooting
For troubleshooting, please visit www.thermofisher.com
References
1. Innis, M.A., et al., PCR Protocols and Applications: A Laboratory Manual, Academic, New York, 1989.
2. Celeda, D., et al., PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ
hybridization, BioTechniques, 12, 98-102, 1992.
3. Finckh, U., et al., Producing single-stranded DNA probes with the
Taq DNA polymerase: a high yield protocol, BioTechniques, 10, 35-39, 1991.
4. Yu, H. et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes, Nucleic Acids Res., 22, 3226-3232, 1994.
5. Innis, M.A., et al., DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-
amplified DNA, Proc. Natl. Acad. Sci. USA, 85, 9436-9440, 1988.
6. Weyant, R.S., et al., Effect of ionic and nonionic detergents on the
Taq polymerase, Biotechniques, 9, 309-308, 1990.
7. Lundberg, K.S., et al., High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus, Gene, 108, 1-
6, 1991.

4 Taq DNA Polymerase (recombinant)

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