T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes. Single-stranded nucleic acids are not substrates for this enzyme.
Applications Cloning (blunt-end or cohesive-end ligation) and adding linkers or adapters to blunt-ended DNA
Source Purified from E. coli lambda lysogen NM989
Performance and quality testing Endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency tested
Unit definition One unit catalyzes the exchange of 1 nmol 32P-labeled pyrophosphate into ATP in 20 min at 37°C. One unit is equal to approximately 300 cohesive-end ligation units.
Unit reaction conditions 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 10 mM DTT, 66 µM ATP, 3.3 µM 32P-labeled pyrophosphate, and enzyme in 0.1 mL for 20 min at 37°C.
For Research Use Only. Not for use in diagnostic procedures.
Contents & storage
T4 DNA Ligase is supplied with a vial of 5X reaction buffer [250 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) polyethylene glycol-8000]. Store at -20°C.