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T4 DNA Ligase

No Product Name Cat.# Conc Size Price(￦) Add
1 T4 DNA Ligase M001S 400 units/μl 20,000 units ￦ 70,000 추가
2 T4 DNA Ligase M001M 400 units/μl 40,000 units ￦ 126,000 추가
3 T4 DNA Ligase M001L 400 units/μl 100,000 units ￦ 280,000 추가
4 T4 DNA Ligase M001H 2000 units/μl 100,000 units ￦ 280,000 추가
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(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

Standard reaction conditions
When a restriction fragment as insert DNA is ligated to a vector DNA.

T4 DNA Ligase (400 unit/μl) 

For sticky ends

For blunt ends

1 μl

2 μl

10X T4 DNA Ligase buffer

2 μl

Vector DNA (50~400 ng/μl)

1 μl

Insert DNA (3X molar excess of vector DNA)

X μl

Distilled water

Up to 20 μl

→ Incubate at room temperature for 1 hr.

 

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.



Figure 1. Detection of nonspecific nuclease activities using radioisotope-labeled DNA substrates

Duplex DNA was prepared by annealing two complementary oligonucleotides (50-nt) and labeled at the 5 ends with -32P ATP by polynucleotide kinase (Cat.# M005). The gel-purified labeled duplex DNA (15 fmol) was then treated with an enzyme to test for the potential contamination of undesired nucleases. In this example, increasing amounts (4,000 to 30,000 units) of T4 DNA Ligase (Cat.#. M001) were incubated with 15 fmol of labeled duplex DNA at 37℃ for 1 hr. Reaction products were analyzed in acrylamide gel electrophoresis, revealing that T4 DNA ligase is free of any nonspecific nuclease activity. , boiled substrate.
　　　　　　　　　　　


Figure 2. Ligation of λ DNA Hind III fragments by T4 DNA Ligase.
Bacteriophage λ DNA (1 μg) was digested with Hind III and then ligated by incubating at 25℃ for 10 min in the presence of increasing amounts of T4 DNA Ligase (Cat.# M001). The amounts of T4 DNA Ligase are as indicated. A significant fraction of ligation products were formed with > 4 units of T4 DNA Ligase. Mw, 1 kb (+) DNA Ladder Marker (Cat.# DM003)



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