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[진행] EZ-Fusion HT Cloning Kit 출시 이벤트 ! ...
[18/07/25]
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[18/07/25]
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[18/07/09]
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[18/07/09]
T4 DNA Ligase
| No | Product Name | Cat.# | Conc | Size | Price(￦) | Add |
|---|---|---|---|---|---|---|
| 1 | T4 DNA Ligase | M001S | 400 units/μl | 20,000 units | ￦ 70,000 |
|
| 2 | T4 DNA Ligase | M001M | 400 units/μl | 40,000 units | ￦ 126,000 |
|
| 3 | T4 DNA Ligase | M001L | 400 units/μl | 100,000 units | ￦ 280,000 |
|
| 4 | T4 DNA Ligase | M001H | 2000 units/μl | 100,000 units | ￦ 280,000 |
|
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.
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)
● Material Safety Data Sheet
- Enzynomics_MSDS_M001_T4 DNA Ligase_E
● Related paper
1. Amangyeld T, et al(2014). Nucl.Acids Res. 42(9), 5846-62
2. Young-Hoon Kang, et al(2009). J Biol Chem. 284, 10376-10386
