T4 DNA Ligase, 400units/μl

(T4 DNA Ligase is a specialized enzyme for cloning.)

Product description
A gene encoding bacteriophage T4 DNA Ligase is cloned and expressed in E. coli, and the recombinanat T4 DNA Ligase is purified to homogeneity. This enzyme catalyzes the formation of a covalent bond between the 5'-phosphate and 3'-OH in nicked duplex DNA or at two DNA ends. This activity is very useful to ligate DNA fragments with either cohesive or blunt ends that are generated by restriction enzyme digestion. T4 DNA Ligase can also ligate RNA with DNA or RNA in a double helix with low efficiency. Single strands of DNA or RNA can not be ligated with T4 DNA Ligase.

Characteristics
- Molecular weight: 55 kDa
- Reaction temperature: Room temperature
- Heat inactivation: 65℃ for 10 min

Applications
- Cloning of restricted DNA fragments into a plasmid
- Addition of linker or adaptor DNAs to cohesive or blund-ended DNA

Quality control
- Purity: >99% on SDS-PAGE
- Endonuclease-free
- Exonuclease-free
- Phosphatase-free

Unit definition
One unit is defined as the amount of enzyme required to ligate 50% of Hind lll digested DNA in a 20 μl reaction mixtures (50 mM Tris-HCl/pH 7.5, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/μl BSA, 0.12 μM 5'-DNA) in 30 min at 16℃. One unit for this cohesive end ligation is equivalent to 0.015 Weiss unit (1 Weiss unit,~66 units).

Storage buffer
10 mM Tris-HCl (pH 7.4), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 200 μg/ml BSA, 50% glycerol

10X T4 DNA Ligase buffer
500 mM Tris-HCl (pH 7.5), 100 mM MgCl₂, 100 mM DTT, 10 mM ATP 

  

Cautions

- The concentration of insert DNA to be cloned must be at least the same or up to 3-fold higher than that of  the vector used.

- T4 DNA Ligase activity is inhibited significantly by NaCl or KCl at 200 mM or a higher concentration.

- The addition of PEG 4000 (final concentration, 5%) can increase significantly ligation efficiencty for blunt-end DNA.

- When the ligated DNA is transformed into E. coli by electroporation, the ligated DNA requires purification by phenol-chloroform treatment followed by ethanol precipitation. The DNA should be dissolved in water for high-efficiency transformation.

- High concentration of DTT (100 mM) in 10X T4 DNA Ligase buffer causes DTT to form precipitates at low temperature. Prior to use, dissolve it completely by warming the buffer. 

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