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 (1). Single-stranded nucleic acids are not substrates for this enzyme. A T4 DNA Ligase Technical Bulletin is available.
Applications: Cloning (blunt-end or cohesive-end ligation) (2). Adding linkers or adapters to blunt-ended DNA (2).
Source: Purified from E. coli œ 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 32 P-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.