In the presence of ATP, T4 DNA ligase forms phosphate between the 3´ hydroxyl and 5´ phosphate termini of double-stranded DNA diester bond. Unique T4 DNA Ligase Buffer optimizes the ligation process and completes the ligation in 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 adapters or adapters to blunt-ended DNA (2).

Source: Purified from Escherichia coliœ prolysin NM989.

Performance and quality testing: Endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease experiments; by ligation efficiency test.

Unit definition: One unit of enzyme can catalyze the passage of 1 nmol ³²P-labeled pyrophosphate in 20 minutes at 37°C. Displacement reaction into ATP. (One unit equals approximately 300 sticky 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, a total of 0.1 ml, react at 37°C for 20 minutes.