T4 DNA Ligase, 5u/ul: Enzyme Description: T4 DNA Ligase catalyzes the formaon of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA or DNA/RNA hybrids, joins DNA fragments with either cohesive or blunt termini. The T4 DNA Ligase requires ATP as a cofactor.
Source: E.coli cells with a cloned gene 30 from bacteriophage T4.
Molecular Weight: 55.3 kDa monomer.
Storage Buffer: The enzyme is supplied in: 20 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% (v/v) glycerol.
10X Reaction Buffer: 400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP (pH 7.8 at 25°C).
50% PEG Solution: 50% (w/v) polyethylene glycol 4000.
Unit Definition: One Weiss unit of the enzyme catalyzes the conversion of 1 nmol of [ 32PPi] into Norit-adsorbable form in 20 min at 37°C (4). One Weiss unit is equivalent to approximately 200 cohesive end ligation units (CEU)*.
Application: Cloning of restriction enzyme generated DNA fragments. Cloning of PCR products. Joining of double-stranded oligonucleotide linkers or adaptors to DNA. Site-directed mutagenesis. Amplified fragment length polymorphism (AFLP). Ligase-mediated RNA detection. Nick repair in duplex DNA, RNA or DNA/RNA hybrids. Self-circularization of linear DNA.
Inhibition and Inactivation: T4 DNA Ligase is strongly inhibited by NaCl or KCl at concentrations higher than 200mM. Inactivated by heating at 65°C for 10 min or at 70°C for 5 min.