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| Product Name | T4 DNA Ligase, Rapid, 600 U/uL, High Concentration |
| Catalog No. | IEC-HMM-0077 |
| Description | High-concentration (600 cohesive end units/uL) T4 DNA ligase for rapid ligation of DNA fragments with cohesive (sticky) or blunt ends in 5-10 minutes at room temperature. The recombinant enzyme catalyzes the formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini in duplex DNA, using ATP as the energy cofactor. The rapid formulation employs an optimized buffer system with PEG 4000 as a macromolecular crowding agent that accelerates the intermolecular ligation rate by 10-100 fold compared to standard T4 ligase reactions. This enzyme is ideal for routine cloning (vector + insert ligation), adapter ligation for NGS library preparation, linker ligation, and DNA circularization, providing >90% ligation efficiency for cohesive ends in 5 minutes and blunt ends in 15-30 minutes. |
| Intended Use | Rapid ligation of double-stranded DNA fragments with cohesive or blunt ends for molecular cloning (plasmid construction, TA cloning, Golden Gate assembly), NGS adapter ligation (Illumina and Ion Torrent library preparation), linker/primer ligation, DNA circularization, and DNA repair. |
| Principle / Technology | T4 DNA ligase catalyzes a three-step reaction: (1) enzyme-AMP adduct formation via ATP (ATP + enzyme -> enzyme-AMP + PPi); (2) AMP transfer to the 5'-phosphate of the donor DNA strand; (3) nucleophilic attack by the 3'-hydroxyl of the acceptor strand on the activated 5'-phosphate, releasing AMP and forming a phosphodiester bond. PEG 4000 in the rapid buffer acts as a macromolecular crowding agent, effectively increasing the local concentration of DNA ends by volume exclusion, dramatically accelerating intermolecular ligation. |
| Detection Method | Mix purified vector DNA (25-50 ng) and insert DNA (3:1 to 10:1 insert-to-vector molar ratio) with 1x Rapid Ligation Buffer and 0.5-1 uL T4 DNA Ligase in 10-20 uL total volume; incubate 5 min at RT (22-25 C) for cohesive ends or 15-30 min at RT for blunt ends; heat-inactivate at 65 C for 10 min if desired (optional — may reduce transformation efficiency); use 1-5 uL for E. coli transformation. |
| Sample Type | Purified double-stranded DNA fragments with 5'-phosphate and 3'-hydroxyl termini; cohesive-end fragments (insert and linearized vector with compatible overhangs of 2-4 bp); blunt-end fragments; PCR products with 3'-A overhang (TA cloning when combined with T-vector). |
| Performance Range / Specifications | Ligation efficiency: >90% cohesive end ligation in 5 min at RT; >80% blunt end ligation in 30 min at RT; cohesive end unit activity: 600 U/uL (1 cohesive end unit = amount ligating 50% of HindIII fragments of lambda DNA in 30 min at 16 C in 20 uL); blunt end unit activity: approximately 60 U/uL. |
| Sensitivity / LOD | Detectable ligation of 1 fmol vector with 3 fmol insert in 10 uL reaction (approximately 10^9 molecules); transformation yield >10^6 CFU/ug pUC19 vector with standard insert ligation. |
| Specificity | T4 DNA ligase specifically catalyzes phosphodiester bond formation at nicks in duplex DNA; requires 5'-phosphate and 3'-hydroxyl termini; does not ligate single-stranded DNA or RNA (though T4 DNA ligase has limited activity on RNA-DNA hybrids and fully duplex RNA under optimized conditions); does not ligate 5'-hydroxyl or 3'-phosphate termini. |
| Reaction Conditions / Protocol | Standard reaction: 1x Rapid Ligation Buffer, vector DNA, insert DNA, 0.5-1 uL T4 DNA Ligase; cohesive ends: 5 min at RT; blunt ends: 15-30 min at RT; heat-inactivate 10 min at 65 C (optional, may reduce transformation efficiency). |
| Components / Formulation | T4 DNA Ligase (600 U/uL in storage buffer: 10 mM Tris-HCl pH 7.4, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol), 2x Rapid Ligation Buffer (132 mM Tris-HCl pH 7.6, 20 mM MgCl2, 2 mM ATP, 2 mM DTT, 15% PEG 4000), Protocol. |
| Storage Conditions | Store at -20 C; avoid repeated freeze-thaw cycles (>20 cycles tolerated); 2x Rapid Ligation Buffer must be completely thawed and mixed before use (PEG precipitation may occur at low temperature — warm to RT and vortex). |
| Shelf Life | 24 months from date of manufacture at -20 C. |
| Package Specifications | 30,000 U (50 uL), 60,000 U (100 uL), 300,000 U (500 uL). |
| Product Form | Liquid; 600 U/uL (cohesive end units) in 50% glycerol storage buffer; 2x Rapid Ligation Buffer supplied separately. |
| Quality Control | Each lot tested: no detectable endonuclease or exonuclease activity (supercoiled plasmid and labeled oligonucleotide assays); ligation efficiency: >90% HindIII lambda fragment ligation in 5 min at RT; transformation efficiency: >1x10^8 CFU/ug pUC19; ATP stability verified after 10 freeze-thaw cycles of buffer. |
| Key Features | 600 U/uL high concentration; rapid 5-min cohesive end ligation; 15-30 min blunt end ligation; PEG 4000 crowding agent; compatible with heat inactivation; high transformation efficiency. |
| Purity | >95% pure by SDS-PAGE; no detectable DNase, RNase, endonuclease, or exonuclease activities. |
| Concentration | 600 cohesive end units/uL; approximately 60 blunt end units/uL; protein concentration approximately 3-5 mg/mL. |
| Activity / Unit Definition | 1 cohesive end unit = amount of enzyme required to ligate 50% of HindIII fragments of lambda DNA (5 ug total) in 30 min at 16 C in 20 uL reaction volume; 1 blunt end unit = amount ligating 50% of HaeIII fragments of PhiX174 DNA (1 ug) in 30 min at 16 C. |
| Molecular Weight | Approximately 55.3 kDa (T4 DNA ligase monomer). |
| Source / Origin | Recombinant bacteriophage T4 DNA ligase (gene 30) expressed in E. coli; purified by multiple chromatography steps; no animal-derived components. |
| pH Range / Optimal pH | Optimal pH 7.5-8.0 (Tris-HCl); 2x Rapid Ligation Buffer pH 7.6; active range pH 6.5-9.0. |
| Shipping Conditions | Cold pack or dry ice; stable at ambient for up to 3 days during transit. |
| Expiration Date / Stability | 24 months at -20 C; quality retained after 20 freeze-thaw cycles; thawed enzyme stable at 2-8 C for 2 weeks; 2x Rapid Ligation Buffer stable at RT for 1 month. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. ISO 9001 certified manufacturing. |
| Compatibility | Compatible with standard molecular cloning workflows; ligation reaction directly transformable into E. coli competent cells without purification (use 1-5 uL per 50 uL competent cells). Compatible with TA cloning using T-vectors. Do not heat-inactivate for electroporation transformation (salts in reaction may cause arcing) — purify ligation by column or ethanol precipitation instead. PEG in the ligation buffer may interfere with some downstream enzymatic reactions if carried over; column purification is recommended before in vitro packaging or other sensitive applications. |
| Recommended Buffer System | 2x Rapid Ligation Buffer: 132 mM Tris-HCl pH 7.6, 20 mM MgCl2, 2 mM ATP, 2 mM DTT, 15% (w/v) PEG 4000. |
| Application Notes / Precautions | Thaw and completely resuspend the 2x Rapid Ligation Buffer before use; PEG 4000 may precipitate at low temperatures — warm to RT and vortex until clear. For optimal blunt-end ligation, use a 5:1 to 10:1 insert:vector molar ratio and 15-30 min incubation. The rapid buffer reduces reaction time from 16 h (16 C) to 5 min (RT) for cohesive ends. For large plasmids (>10 kb), increase incubation time to 15-30 min for cohesive ends. The ligation reaction can be used directly for chemical transformation (1-5 uL) but should be purified before electroporation. The heat inactivation step (65 C, 10 min) inactivates the enzyme but may reduce transformation efficiency by 20-50%; it can be omitted for chemical transformation. |
| Batch-to-Batch Consistency | Cohesive end ligation efficiency >90% for all lots; transformation efficiency >1x10^8 CFU/ug pUC19 for all lots; activity 600 +/-60 U/uL. |
For research use only, not for clinical use.
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