Description:
Genscript T4 DNA Ligase is prepared from E. coli carrying a cloned gene 30 of bacteriophage T4. T4 DNA ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5’-phosphate and 3’-hydroxyl termini in duplex DNA or RNA with blunt or cohesive-end termini. GenScript T4 DNA Ligase repairs single-strand nicks in duplex DNA, RNA, and DNA-RNA hybrids but has no activity on single-stranded nucleic acids. All T4 DNA ligase is inactivated by heating at 65°C for 10 minurtes.

Applications:

• Cloning of restriction fragments
• Joining linkers and adapters to blunt-ended DNA.

Contents:

• T4 DNA Ligase
• 10X T4 DNA Ligase reaction buffer: 500 mM Tris-HCl, 100 mM MgCl₂, 5 mM ATP, 100 mM Dithiothreitol, 250 µg/ml BSA, pH 7.5 at 25°C

Quality Control:
Purified free of contaminating endonucleases and exonucleases. Each lot of T4 DNA Ligase is also tested in a mock cloning assay.

Unit definition:
One unit is defined as the amount of enzyme required to give 90% ligation of 6 µg Hind III fragments of λ DNA in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer.

Storage Buffer and Concentration:
The product is supplied in 400 U/µl in a 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 200 µg/ml BSA, 50% glycerol, pH 7.4 at 25°C.

Storage:
Store the product at -20°C. It will remain stable for at least one year if stored properly.

Note:
1. One unit is equivalent to approximately 0.008 Weiss Unit. Weiss Unit activity assay: 66 mM Tris-HCl (pH7.6), 6.6 mM MgCl₂, 10 mM DTT, 66 μM ATP, 3.3 μM [³²P] Na₄P₂O₇.
2. Requirements: Mg²⁺, ATP and DTT. The optimum concentration of Mg²⁺ is 10 mM. Mn²⁺ may be substituted for Mg²⁺ but is only 25% as effective as Mg²⁺.
3. Inhibition: 50% inhibition by greater than 150 mM NaCl (activity measured at nicks). Other inhibitors include 0.2 M K⁺, Cs⁺, Li⁺, NH⁴⁺ and 1 mM spermine.
4. It is necessary to remove the enzyme from the ligation mixture by chloroform extraction prior to electro-transformation of bacterial cells with DNA.

PROTOCOL:  20070209010256  (PDF)
MSDS:  20080228032803  (PDF)