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Product Contents

Taq DNA Polymerase in Storage Buffer A:

Note: Cat.# M1861, M1865 and M1868 are supplied with Thermophilic DNA Polymerase 10X Buffer and a separate tube containing MgCl2. Cat.# M2861, M2865 and M2868 are supplied with Taq DNA Polymerase 10X Buffer, which contains 15mM MgCl2.

Important! Taq DNA Polymerase in Storage Buffer A must be used with the Reaction Buffer provided. If another reaction buffer is used, Triton® X-100 must be added to a final concentration of 0.1% to ensure enzyme activity.

Taq DNA Polymerase in Storage Buffer B:

Note: Cat.# M1661, M1665 and M1668 are supplied with Thermophilic DNA Polymerase 10X Buffer and a separate tube of MgCl2. Cat.# M2661, M2665 and M2668 are supplied with Taq DNA Polymerase 10X Buffer, which contains 15mM MgCl2.

Taq DNA Polymerase in Storage Buffer B is fully compatible with other reaction buffers.

Description: Taq DNA Polymerase(a) is a thermostable enzyme that replicates DNA at 74°C and exhibits a half-life of 40 minutes at 95°C (1,2). Taq catalyzes the polymerization of nucleotides into duplex DNA in the 5′→ 3′ direction in the presence of magnesium. The enzyme has a molecular weight of 94,000 daltons as estimated from the predicted amino acid sequence and exhibits 5′→ 3′ exonuclease activity. Taq is recommended for use in PCR and primer extension reactions at elevated temperatures.

Enzyme Storage Buffer A: The composition of Storage Buffer A is 50mM Tris-HCl (pH 8.0 at 25°C), 100mM NaCl, 0.1mM EDTA, 1mM DTT, 50% glycerol and 1% Triton® X-100.

Enzyme Storage Buffer B: The composition of Storage Buffer B is 20mM Tris-HCl (pH 8.0 at 25°C), 100mM KCl, 0.1mM EDTA, 1mM DTT, 50% glycerol, 0.5% Tween® 20 and 0.5% Nonidet®-P40.

Source: Thermus aquaticus strain YT1 (1).

Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nanomoles of dNTPs into acid insoluble material in 30 minutes at 74°C (2). The reaction conditions are specified below, under Standard DNA Polymerase Assay Conditions.

Thermophilic DNA Polymerase 10X Buffer, Magnesium Free (Part# M190A, M190G): When the 10X Buffer supplied with this enzyme is diluted 1:10 it has a composition of 10mM Tris-HCl (pH 9.0 at 25°C), 50mM KCl and 0.1% Triton® X-100. This buffer is optimized for use with 200µM each of dNTPs. Sufficient 25mM MgCl2 is provided separately to allow optimization of enzyme performance under a variety of conditions. The Triton® X-100 in the Buffer is compatible with the detergents in the Storage Buffer of the enzyme for all applications.

Taq DNA Polymerase 10X Buffer containing 15mM MgCl2 (Part# M188A, M188J): This buffer, which has a composition of 50mM KCl, 10mM Tris-HCl (pH 9.0 at 25°C), 1.5mM MgCl2 and 0.1% Triton® X-100 when diluted 1:10, is supplied with Cat.# M2661, M2665, M2668 and M2861, M2865, M2868.

Magnesium Chloride Solution, 25mM (Part# A351B, A351H): The final magnesium concentration in a reaction mixture may be optimized by the user according to individual requirements. It is important to vortex the MgCl2 thoroughly after thawing and prior to use to disperse the MgCl2 throughout the tube.

Storage Temperature: Store at –20°C. Avoid exposure to frequent temperature changes. See the expiration date on the Product Information Label.

Quality Control Assays

Activity Assays

Functional Assay: Taq DNA Polymerase is tested for performance in the polymerase chain reaction (PCR) using 1.25 units of enzyme to amplify a 360bp region of the α -1-antitrypsin gene from 100 molecules (0.35ng) of human genomic DNA. The resulting PCR product is visualized as a single band on an ethidium bromide-stained agarose gel.

Standard DNA Polymerase Assay Conditions (not PCR conditions): The polymerase activity is assayed in 50mM Tris-HCl (pH 9.0), 50mM NaCl, 5mM MgCl2, 200µM each of dATP, dGTP, dCTP, dTTP (a mix of unlabeled and [3H]dTTP), 10µg activated calf thymus DNA and 0.1mg/ml BSA in a final volume of 50µl. The test result is listed on the Product Information Label.

Contaminant Assays

Endonuclease Assay: One microgram of supercoiled plasmid DNA is incubated with 5 units of Taq DNA Polymerase for 8 hours at 45°C and 8 hours at 70°C in 1X Thermophilic DNA Polymerase Reaction Buffer and 1.5mM MgCl2. Following incubation, the DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking.

Exonuclease Assay: One microgram of λ DNA and 1µg of λ /Hind III DNA are incubated with 5 units of Taq DNA Polymerase for 8 hours at 45°C and 8 hours at 70°C in 1X Thermophilic DNA Polymerase Reaction Buffer and 1.5mM MgCl2. Following incubation, the DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible smearing.

Physical Purity: Taq DNA Polymerase is determined to be >90% pure as judged by SDS-polyacrylamide gels with Coomassie® blue staining.

Part# 9PIM166

Revised 12/04

AF 9 PI M1 6 6 1 2 0 4 M1 6 6

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Part# 9PIM166

Printed in USA. Revised 12/04

Taq DNA Polymerase

M1861, M1865, M1868 and M2861, M2865, M2868

M1661, M1665, M1668 and M2661, M2665, M2668

Storage Buffer Compatibility with Reaction Buffers

Promega Taq DNA Polymerase in Storage Buffer A (products with catalog numbers M1861, M1865, M1868 and M2861, M2865, M2868) must be used with the provided Promega Reaction Buffer. Use of other reaction buffers that do not contain Triton® X-100 will result in inactivation of the enzyme. If the user prefers another reaction buffer, Triton® X-100 must be added to a final concentration of 0.1% to ensure enzyme activity.

Promega Taq DNA Polymerase in Storage Buffer B (products with catalog numbers M1661, M1665, M1668 and M2661, M2665, M2668) is fully compatible with other reaction buffers.

Magnesium Concentration Effects in PCR

The concentration of magnesium used in PCR is critical to the success of the reaction. The following steps should be taken to ensure optimal magnesium concentration:

•Allow the MgCl2 solution to thaw completely before using.

•Vortex the MgCl2 solution after thawing.

•Titrate MgCl2 between 1.0 and 3.5mM in 0.5mM increments in order to identify optimal MgCl2 concentration.

Figure 1. Demonstration of buffer compatibility. Amplification by PCR of a 360bp region of the human α -1-antitrypsin gene. Lanes 4 and 8: 100bp DNA Ladder (Cat.# G2101). Lanes 1–3; 5–7: PCR products.

Figure 2. Demonstration of effects of magnesium concentration on PCR amplification. Lane M: pGEM® DNA Markers (Cat.# G1741). Lanes 2–9: Amplification of a 1.8kb luciferase gene product.

References

1.Chien, A., Edgar, D.B. and Trela, J.M. (1976) Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.

J. Bacteriol. 127, 1550–7.

2.Kaledin, A.S., Sliusarenko, A.G. and Gorodetskii, S.I. (1980) Isolation and properties of DNA polymerase from extreme thermophylic bacteria

Thermus aquaticus YT-1. Biokhimiia 45, 644–51.

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Product must be within expiration date and have been stored and used in accordance with product literature. See Promega Product Insert for specific tests performed.

Part# 9PIM166

Printed in USA. Revised 12/04