T4 DNA Ligase, 5u/ul: T4 DNA Ligase, 5u/ul
Enzyme Description:
T4 DNA Ligase catalyzes the formation 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.
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.
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. One Weiss unit is equivalent to approximately 200 cohesive end ligation units (CEU)*.
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.
