- Home
- IVD
- By Technology Types
- By Diseases Types
- By Product Types
- Research
- Resource
- Distributors
- Company
| Product Name | T4 DNA Ligase |
| Catalog No. | IEC-HMM-0056 |
| Description | Bacteriophage T4 DNA ligase catalyzes the ATP-dependent formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini in double-stranded DNA. This enzyme joins both cohesive (sticky) and blunt-ended DNA fragments with high efficiency and is the standard ligase for molecular cloning applications. |
| Intended Use | For DNA fragment ligation in molecular cloning, adapter ligation for NGS library preparation, linker ligation, double-stranded oligonucleotide circularization, and nick-sealing in double-stranded DNA substrates. |
| Principle / Technology | ATP-dependent DNA ligation proceeds through a three-step mechanism: (1) auto-adenylation of a lysine residue in the enzyme active site using ATP, forming an enzyme-AMP covalent intermediate; (2) transfer of AMP to the 5'-phosphate of the donor DNA strand; (3) nucleophilic attack by the 3'-OH of the acceptor strand, displacing AMP and forming the phosphodiester bond. |
| Detection Method | One Weiss unit catalyzes the exchange of 1 nmol ³²P from pyrophosphate into Norit-adsorbable material in 20 min at 37 °C. Cohesive-end units (CEU) measured by ligation of HindIII-digested lambda DNA fragments. |
| Sample Type | Purified double-stranded DNA fragments with compatible cohesive or blunt ends. Quantities ranging from femtomoles (NGS adapters) to picomoles (cloning vectors). |
| Performance Range / Specifications | Ligation efficiency: >95% circularization of cohesive-end fragments. Blunt-end ligation requires approximately 50-fold more enzyme or longer incubation. Broad temperature optimum: 4-25 °C. |
| Sensitivity / LOD | Detectable ligation of 1 fmol cohesive-end fragments in 10 uL reaction after 1 hour at 16 °C. |
| Specificity | Specific for double-stranded DNA. Minimal activity on single-stranded DNA or RNA substrates. Requires 5'-phosphate on donor strand. |
| Reaction Conditions / Protocol | Vector (50-100 ng) + insert (3:1 to 5:1 molar ratio) + 1 uL T4 DNA Ligase (400 U/uL) + buffer. Incubate 16 °C 1-4 h (cohesive) or 4-16 h (blunt). Heat inactivate at 65 °C 10 min before transformation. |
| Components / Formulation | T4 DNA Ligase (400 U/uL cohesive-end, or 1 U/uL Weiss) in 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol. Supplied with 10x T4 DNA Ligase Buffer containing ATP. |
| Storage Conditions | Store at -20 °C to -30 °C. ATP in the 10x buffer is labile; prepare single-use buffer aliquots. |
| Shelf Life | 24 months at -20 °C. |
| Package Specifications | 20,000 U (50 uL), 100,000 U (250 uL), 500,000 U (1.25 mL). Includes 10x Ligation Buffer. |
| Product Form | Clear, colorless glycerol-based liquid. |
| Quality Control | Each lot tested for: cohesive-end ligation activity; blunt-end ligation activity; absence of contaminating exonuclease and endonuclease; functional cloning performance (transformation efficiency of ligated products). |
| Key Features | Industry-standard cloning ligase; high specific activity; performs both cohesive-end and blunt-end ligation; active at low temperatures (4-25 °C) allowing optimization for fragment stability. |
| Purity | >= 95% by SDS-PAGE. No detectable exo/endonuclease activities. |
| Concentration | 400 cohesive-end units/uL (approximately 1 Weiss unit/uL). High-concentration variant (2,000 U/uL) available for NGS library preparation. |
| Activity / Unit Definition | 1 cohesive-end unit ligates >90% of HindIII fragments of lambda DNA (5'-cohesive termini) in 30 min at 16 °C. 1 Weiss unit = 1 nmol ³²P-pyrophosphate exchanged in 20 min at 37 °C. |
| Molecular Weight | ~55 kDa (monomer). Active as monomer. |
| Source / Origin | Recombinant enzyme produced in E. coli from bacteriophage T4 gene 30. |
| pH Range / Optimal pH | Optimal pH 7.2-7.8. Active range pH 6.5-8.5. |
| Shipping Conditions | Shipped with cold packs. ATP in buffer is freeze-thaw sensitive; aliquot buffer upon receipt. |
| Expiration Date / Stability | 24 months at -20 °C. 10x buffer: stable 12 months at -20 °C; aliquot and avoid repeated freeze-thaw cycles to preserve ATP integrity. |
| Regulatory / Compliance | ISO 9001 certified manufacturing. Suitable for IVD raw material supply. |
| Compatibility | Compatible with standard molecular biology-grade water, TE buffer, and common restriction enzyme digestion buffers after heat inactivation of restriction enzymes. Not compatible with buffers containing >50 mM NaCl or phosphate. |
| Recommended Buffer System | 10x Ligation Buffer: 500 mM Tris-HCl (pH 7.5), 100 mM MgCl₂, 10 mM ATP, 100 mM DTT. Rapid ligation buffer variants available for 5-minute room temperature ligations. |
| Application Notes / Precautions | For cohesive-end ligation: use 1-3:1 insert:vector molar ratio. For blunt-end ligation: use 5-10:1 ratio and increase enzyme to 5 U per reaction. Dephosphorylate vector with alkaline phosphatase to reduce self-ligation background. Heat-inactivation at 65 °C for 10 min or purify ligation products before electroporation. |
| Batch-to-Batch Consistency | Lot-to-lot ligation efficiency variation <= +/- 15% as measured by transformation colony counts of standardized ligation reactions. |
For research use only, not for clinical use.
|
There is no product in your cart. |
Copyright © 2026 Alta DiagnoTech. All rights reserved.
