T4 DNA ligase uses the energy of ATP to catalyze the formation of phosphate from the 3´ hydroxyl end and the 5´ phosphate end between double-stranded DNA diester bond. This unique T4 DNA ligase buffer optimizes ligation reactions to complete in less than 5 minutes (1). Single-stranded nucleic acids are not substrates for this enzyme. Provides T4 DNA Ligase technology bulletin.

Applications: Cloning (blunt or sticky end ligation) (2). Add linkers or adapters to blunt-ended DNA (2).

Source: Purified from Escherichia coliœ prolysin NM989.

Performance and quality testing:Endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease assay; tested for ligation efficiency.

Unit definition: One unit of enzyme can catalyze the conversion of 1 nmol ³²P-labeled pyrophosphate into ATP. (One unit is equivalent to approximately 300 sticky end ligation units.)

Unit reaction conditions:66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl_{2< /sub>, 10 mM DTT, 66 µM ATP, 3.3 µM 32P-labeled pyrophosphate and 0.1 mL enzyme, 37°C for 20 min.}