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| Product Name | T4 DNA Ligase and Rapid Ligation Kit |
| Catalog No. | NATR-HMM-0130 |
| Description | A complete kit for the efficient ligation of DNA fragments, containing high-concentration T4 DNA ligase with an optimized rapid ligation buffer. The formulation enables cohesive-end ligations in 5 minutes and blunt-end ligations in 15 minutes, significantly accelerating the standard cloning workflow. |
| Intended Use | Ligation of DNA fragments with cohesive (sticky) or blunt ends into plasmid, cosmid, or bacteriophage vectors for molecular cloning; linker and adapter addition to DNA fragments for next-generation sequencing library preparation; and DNA repair and circularization applications. |
| Principle / Technology | T4 DNA ligase catalyzes the ATP-dependent formation of phosphodiester bonds between adjacent 3ʹ-hydroxyl and 5ʹ-phosphate termini in double-stranded DNA. The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids but does not ligate single-stranded nucleic acids. The rapid ligation buffer provides optimized conditions including PEG to accelerate intermolecular ligation reactions. |
| Detection Method | Transformation of ligation products into competent E. coli, followed by colony counting (efficiency); colony PCR or restriction digestion of plasmid DNA from transformants (accuracy); agarose gel electrophoresis to verify ligation product size shift |
| Sample Type | DNA fragments with 5ʹ-phosphate and 3ʹ-hydroxyl ends: cohesive-end restriction fragments, blunt-end fragments, PCR products with 3ʹ-A overhangs (with compatible T-vector), synthetic adapters and linkers |
| Performance Range / Specifications | Ligation time: 5 minutes at room temperature for cohesive ends, 15 minutes at room temperature for blunt ends (rapid protocol); 16 hours at 4°C or 16°C for maximum efficiency (overnight protocol); insert-to-vector molar ratio optimization range: 1:1 to 10:1 |
| Sensitivity / LOD | Cohesive-end ligation: >95% of vector molecules recircularize with insert in optimal conditions; blunt-end ligation: >70% efficiency; transformation background ≤5% (vector self-ligation without insert) with appropriate phosphatase treatment |
| Specificity | Sequence-accurate ligation verified by Sanger sequencing of recombinant clones (>98% correct junctions); minimal deletions, insertions, or rearrangements at ligation boundaries |
| Reaction Conditions / Protocol | Set up 20 μL reaction: vector DNA (20–100 ng), insert DNA (3:1 to 5:1 molar ratio to vector), 2 μL 10× rapid ligation buffer, 1 μL T4 DNA ligase (400 U/μL), nuclease-free water to volume; mix by pipetting, incubate at room temperature: 5 minutes (cohesive), 15 minutes (blunt), or 16 hours at 16°C (overnight); use 2–5 μL for chemical transformation or 1 μL for electroporation; optional heat inactivation at 65°C for 10 minutes |
| Components / Formulation | T4 DNA ligase (400 U/μL and 2,000 U/μL high-concentration option, E. coli recombinant), 10× rapid ligation buffer (Tris-HCl, MgCl2, DTT, ATP, PEG 4000 or PEG 8000), nuclease-free water |
| Storage Conditions | Enzyme and buffer stored at –20°C; enzyme must be kept on ice when in use; buffer should be thawed, vortexed thoroughly to dissolve any PEG precipitate, and kept on ice; ATP in buffer is freeze-thaw sensitive — aliquot buffer into single-use volumes |
| Shelf Life | 18 months at –20°C from date of manufacture |
| Package Specifications | 100 reactions and 500 reactions; includes T4 DNA ligase (400 U/μL), 10× rapid ligation buffer, and nuclease-free water |
| Product Form | Liquid enzyme in storage buffer (50% glycerol, 10 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100); liquid 10× buffer |
| Quality Control | Each lot tested: >95% cloning efficiency for cohesive-end ligation of HindIII-digested vector with 3:1 insert ratio; >70% for blunt-end ligation; background <5% (vector-only control); sequence accuracy >98% (Sanger verification of 10 random clones); no detectable exonuclease or endonuclease contamination |
| Key Features | Ultra-fast 5-minute cohesive-end ligation saves hours; high-concentration enzyme option for challenging blunt-end and large-fragment clone; PEG-enhanced buffer drives rapid intermolecular ligation; validated for Gibson Assembly-compatible fragment preparation; active in restriction enzyme buffers for one-step restriction-ligation protocols |
For research use only, not for clinical use.
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