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 16oC 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 (25oC) should be stored at -20oC.
Heat Inactivation:
65oC for 10 minutes
1X T4 DNA Ligase Reaction Buffer:
50mM Tris-HCl, 10mM MgCl2, 1mM ATP, 10mM dithiothreitol, 25µg/ml BSA, pH 7.5 (25oC)
Reaction Conditions:
1X T4 DNA Ligase Reaction Buffer, incubate at 16oC.
For convenience, ligations may be done at room temperature (20-25oC). 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.
