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DNA Modifying Enzymes and Cloning >
Ligases >
Taq DNA Ligase |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Thermostable ligase for incorporation of phosphorylated oligonucleotides during PCR and Ligase Chain Reaction
- Taq DNA Ligase is NOT a substitute for T4 DNA Ligase
- Supplied with 10X Reaction Buffer containing NAD+
Description:
Taq DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini of two adjacent oligonucleotides which are hybridized to a complementary target DNA. The ligation will occur only if the oligonucleotides are perfectly paired to the complementary target DNA and have no gaps between them; therefore, a single-base substitution can be detected. Taq DNA Ligase is active at elevated temperatures (45°C-65°C) (1,2).
Source:
Purified from an E. coli strain containing the cloned ligase gene from Thermus aquaticus HB8 (1).
Applications:- Allele-specific gene detection using Ligase Detection Reaction and Ligase Chain Reaction (1,3).
- Mutagenesis by incorporation of a phosphorylated oligonucleotide during primer extension amplification (4).
Reagents Supplied:
λ DNA-BstEII Digest
Taq DNA Ligase Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
No
Specific Activity:
1,670,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X Taq DNA Ligase Reaction Buffer
Incubate at
45°C.
1X Taq DNA Ligase Reaction Buffer:
20 mM Tris-HCl
25 mM potassium acetate
10 mM Magnesium Acetate
1 mM NAD
10 mM Dithiothreitol
0.1 % Triton X-100
pH 7.6 @ 25°C
Unit Definition:
(Cohesive End Unit)
One unit is defined as the amount of enzyme required to give 50% ligation of the 12-base pair cohesive ends of 1 µg of BstEII-digested λ DNA in a total reaction volume of 50 µl in 15 minutes at 45°C.
Unit Assay Conditions: 1X Taq DNA Ligase Reaction Buffer and DNA (20 µg/ml). After incubation at 45°C for 15 minutes, the reaction is terminated by addition of stop dye (50% glycerol, 50 mM EDTA and bromophenol blue), heated at 70°C for 10 minutes and then loaded on a 0.7% agarose gel. Due to the presence of ligase, the cos ends of BstEII-digested λ DNA will stay together after 70°C heat treatment.
Concentration:
40,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Reaction Conditions: Incubate DNA and enzyme in 1X Taq DNA Ligase Buffer at 45°C for 15 minutes or in a thermocycler with a program suited to the reaction described by Barany (1991) Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase. Proc. Natl. Acad. Sci. USA 88, 189-193. The reaction is stopped with a mixture of 50% glycerol, 50 mM EDTA, bromphenol blue."
1X Taq DNA Ligase Reaction Buffer requires NAD+ as a cofactor. NAD+ is supplied in the 10X Taq DNA Ligase Reaction Buffer; the buffer should be stored at -70°C to extend the half life of the NAD+ cofactor.
FAQs
- Is Taq DNA Ligase used for a special technique?
- How much ligation occurs at mismatches when using Taq DNA Ligase?
- How many temperature cycles will Taq DNA Ligase survive?
- Can Taq DNA Ligase be used for cloning?
- What is the molecular weight of Taq DNA Ligase?
- What is the specific activity of Taq DNA Ligase?
- Why is the Taq DNA Ligase buffer brown?
- Does Taq DNA Ligase require NAD?
- What is the activity of Taq DNA Ligase in other NEBuffers?
- What is the activity of Taq DNA Ligase at various temperatures?
- What is the stability of Taq DNA Ligase at 95°C?
- What is the stability of Taq DNA Ligase at room temperature?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Each lot is tested for contaminating single-stranded DNA exonuclease, endonuclease, ribonuclease and phosphatase activities.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 1,200 units of Taq DNA Ligase with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 1,500 units of Taq DNA Ligase with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Nuclease Activity:
Incubation of 4,000 units for 16 hours at 37°C in the recommended assay buffer plus NAD with Sma I-Sal I fragments of λ DNA does not alter the expected banding pattern on agarose gels. Incubation of Hind III fragments of λ DNA with 80 units of enzyme for 16 hours at 37°C in the recommended assay buffer without NAD does not alter the λ Hind III banding pattern on agarose gels.
References
- Barany, F. (1991) Proc. Natl. Acad. Sci. USA, 88, 189-193.
- Takahashi, M. et al. (1984) J. Biol. Chem., 259, 10041-10047.
- Barany, F. (1991) The Ligase Chain Reaction in a PCR World, 5-16.
- Michael, S.F. (1994) Biotechniques, 16, 411-412.
Reagents Sold Separately
λ DNA-BstEII Digest
Taq DNA Ligase Reaction Buffer
Legal
Research Use Assurance:
RESEARCH USE ONLY - No License to Use in Any Patented Process Granted or Implied
Notice to Purchaser about Limited License
This product is designed to ligate DNA fragments at temperatures requiring a thermoactive and thermostable enzyme. The seller is aware that the product may be used in the Ligase Chain Reaction (LCR™) process covered by one or more claims of a pending patent application or issued patent assigned to Cornell Research Foundation, Inc. or Cornell Research Foundation, Inc. and the California Institute of Technology. The seller has a limited license under such patent rights, and purchase of this product includes a fully paid-up limited, non-exclusive sublicense under such intellectual property rights to use this product to perform the Ligase Chain Reaction™ process only for research and development activities and for industrial quality assurance testing. No other license has been granted expressly, impliedly or by estoppel to seller, and seller grants no other license or sublicense expressly, impliedly or by estoppel.
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