| Description | T3 DNA Ligase is an ATP-dependent ds DNA ligase from bacteriophage T3. It catalyzes the formation of a phosphodiester bond between a 5′ phosphate and a 3′ hydroxyl termini in duplex DNA. T3 DNA Ligase can efficiently catalyze ligation of both cohesive ends and blunt ends and nick sealing. T3 DNA Ligase is 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but not as active as T4 DNA ligase for blunt-ended DNA fragments, although the blunt end ligation can be enhanced by the addition of 15% PEG 6000 to the reaction. T3 DNA Ligase displays a higher efficiency for joining A/T overhangs than C/G matched ends, therefore it can be used for TA cloning. Cohesive ligation with T3 DNA Ligase can be completed in less than 10 min. |
| Applications | - Cloning of DNA fragments generated by restriction enzyme digestion
- Adding linkers or adapters to dsDNA
- Circularization of linear DNA
- Nick-sealing in dsDNA
- Site-directed mutagenesis
- Golden Gate assembly of DNA fragments such as Transcription Activator-Like Effector Nucleases (TALEN)
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| Source | Purified from a recombinant E. coli strain carrying the T3 DNA Ligase gene |
| Components | - T3 DNA Ligase: 3,000,000 U/ml in storage buffer: 20 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol, pH 7.5 @ 25°C .
- 4X T3 DNA Ligation Buffer: 250 mM Tris-HCI, 40 mM MgCl2, 4 mM DTT, 4 mM ATP, 30% PEG 6000, pH 7.6 @ 25°C
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| Unit Definition | 1 unit is defined as the amount of T3 DNA Ligase required to ligate 50% of 100 ng HindIII DNA fragments with cohesive termini in 30 minutes at 23°C |
| Quality Control | - The absence of endo-, exodeoxyribonucleases, phosphatases, and ribonucleases confirmed by appropriate quality tests.
- Functionally tested for the capacity to join cohesive- and blunt-end DNA fragments.
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| Storage | -20°C. |
