Tth DNA Ligase

Cat. No.	Concentration	Size
SRE00037-1	5,000 units/ml	250 units
SRE00037-2	5,000 units/ml	1,250 units

Description

Tth DNA Ligase is a thermostable DNA ligase derived from Thermus thermophilus that catalyzes the ATP-dependent formation of phosphodiester bonds between adjacent 3’-OH and 5’-phosphate termini in double-stranded DNA. The enzyme retains full activity at elevated temperatures, providing high specificity for perfectly matched base pairs at the ligation junction. It is ideally suited for ligase chain reaction (LCR), SNP genotyping, and other high-stringency ligation applications that require thermal cycling.

Features

Thermostable – Optimal activity at 55–65°C; remains active after repeated heating.
High Specificity – Strong mismatch discrimination at the ligation site.
ATP-Dependent – Requires ATP and Mg²⁺ as cofactors.
Thermal Cycling Compatible – Suitable for LCR and other temperature-cycled ligation protocols.
Ideal for Diagnostics – Perfect for mutation detection and genotyping assays.

Applications

Ligase Chain Reaction (LCR) for SNP/mutation detection
High-temperature blunt- or cohesive-end DNA ligation
Gap-filling and nick sealing at elevated temperatures
High-stringency adapter ligation in NGS workflows
DNA repair and modification studies under thermophilic conditions

Unit Definition

One unit is defined as the amount of enzyme required to ligate more than 50% of 100 ng of HindIII-cut λ DNA fragments in 30 minutes at 45°C.

Storage Temperature

-20°C for optimal long-term stability

Storage Buffer

25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 50% Glycerol

Specifications

Expression System: Escherichia coli
Theoretical Molecular Weight: ~74,000 Daltons
Concentration: 5,000 units/ml
Heat Inactivation: No
Cofactor: ATP (0.5–1 mM)
Optimal Temperature: 45–65°C

Quality Control

Each lot undergoes comprehensive testing including:

Functional activity assays
Purity analysis by SDS-PAGE (>95%)
Endonuclease, exonuclease, DNase and DNA contamination testing
Sterility verification

Datasheet

MSDS

Important Handling Notes:

Avoid repeated freeze-thaw of the enzyme.
Store at –20°C for long-term stability.
Thaw on ice before use
Requires both ATP and Mg²⁺ for activity.
Higher temperatures increase mismatch discrimination, reducing off-target ligation.
Works with blunt and cohesive ends, though blunt-end ligation may require PEG or extended incubation.
Not recommended for RNA ligation.
Compatible with thermal cycling protocols for mutation detection.

Frequently Asked Questions

Q1: How is Tth DNA Ligase different from T4 DNA Ligase?
A: Tth DNA Ligase is thermostable and designed for high-stringency ligation at elevated temperatures, while T4 DNA Ligase works best at low/moderate temperatures and is more versatile for routine cloning.

Q2: Can Tth DNA Ligase be used in LCR?
A: Yes, it is the preferred choice for LCR due to its thermostability and mismatch discrimination.

Q3: Can it ligate blunt ends?
A: Yes, but efficiency is lower than cohesive-end ligation; addition of PEG can improve performance.

Troubleshooting

Problem: Low ligation efficiency

- Verify ATP and Mg²⁺ are present and fresh.
- Use optimal temperature (55–65°C).
- Ensure DNA ends are fully base-paired and phosphorylated.

Problem: Nonspecific ligation

- Increase reaction temperature to improve specificity.
- Reduce ligase amount or shorten ligation time.

Disclaimer

Research Applications Only
This product is intended for research use or further manufacturing purposes only. Not for diagnostic procedures or direct therapeutic applications.

References

Barany F. Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci USA. 1991;88(1):189–193.

Wiedmann M, Wilson WJ, Czajka J, Luo J, Barany F, Batt CA. Ligase chain reaction (LCR)—overview and applications. PCR Methods Appl. 1994;3(4):S51–S64.

Luo J, Bergstrom DE, Barany F. Improving the fidelity of thermostable ligases with low-molecular-weight additives. Nucleic Acids Res. 1996;24(14):3071–3078.