T4 DNA Ligase datasheet

T4 DNA ligase
(Cat. No.: E003)

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:
    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^oC in 1X T4 DNA Ligase Reaction Buffer.

Concentration:
    400,000 units/ml

Quality Control Tests:
    Activity, SDS-PAGE (purity), DNase, endonuclease/nickase, RNase.

Storage:
    T4 DNA ligase in 10 mM Tris-HCl, 50mM KCl, 1mM dithiothreitol, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol, pH 7.4 (25^oC) should be stored at -20^oC.

Heat Inactivation:
    65^oC for 10 minutes

1X T4 DNA Ligase Reaction Buffer:
    50mM Tris-HCl, 10mM MgCl₂, 1mM ATP, 10mM dithiothreitol, 25µg/ml BSA, pH 7.5 (25^oC)

Reaction Conditions:
    1X T4 DNA Ligase Reaction Buffer, incubate at 16^oC.
    For convenience, ligations may be done at room temperature (20-25^oC). 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.

References:
    Engler, M.J. et al. (1982) P.D. Boyer (Eds.), The Enzymes, 5, pp. 3. San Diego: Academic Press.
    Remaut, E. et al. (1983) Gene, 22, 103-113.
    Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 1.53-1.73.
    Weiss, B. et al. (1968) J. Biol. Chem., 243, 4543-4555.