T4 DNA ligase

• Cat.No.: CMC12

Specification

• Cat. No.: CMC12
• Description: T4 DNA Ligase is a recombinant enzyme formulated and tested for high activity in blunt- and cohesive-end ligations for cloning and linker addition. The enzyme covalently joins 5"-phosphate and 3"-hydroxyl termini in duplex DNA or RNA. It is free of detectable exo- or endonuclease activities.
• Source: Purified from recombinant E. coli strain.
• Applications: Cloning of restriction fragments; Joining linkers and adapters to blunt-ended DNA.
• Unit Definition (Cohesive End Ligation Unit): One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of lambda DNA (5" DNA termini concentration of 0.12μM, 300μg/ml) in a total reaction volume of 20μl in 30 minutes at 16°C in 1× T4 DNA Ligase Reaction Buffer. One Cohesive End Ligation Unit equals 0.015 Weiss (ATP-PP exchange) units, or one Weiss unit equals 67 Cohesive End Ligation Units.
• Concentration: 400,000 units/ml.
• Quality Control Tests: Activity, SDS-PAGE (purity), DNase, endonuclease/nickase, RNase.
• Storage: T4 DNA ligase in 10mM Tris-HCl, 50mM KCl, 1mM dithiothreitol, 0.1mM EDTA, 0.2 mg/ml BSA, 50% glycerol, pH 7.4 (25℃) should be stored at -20°C.
• Heat Inactivation: 65°C for 10 minutes.
• 1× T4 DNA Ligase Reaction Buffer: 50mM Tris-HCl, 10mM MgCl2, 1mM ATP, 10mM dithiothreitol, 25μg/ml BSA, pH7.5 (25℃)).
• Reaction Conditions: 1× T4 DNA Ligase Reaction Buffer, incubate at 16°C. For convenience, ligations may be done at room temperature (20-25°C). For cohesive (sticky) ends, use 1μl of T4 DNA Ligase in a 20μl reaction for 10 minutes. For blunt ends, use 1μl of T4 DNA Ligase in a 20μl reaction for 2 hours.