T4 DNA Ligase catalyzes the formation of a phosphodiester bonds between 5’ phosphate and 3’ hydroxyl termini in duplex DNA/RNA. This enzyme can join-blunt end and cohesive-end termini, repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.

Concentration

50 – 100 units/μl

Source

Purified from E. coli strain harbouring the plasmid that directs the synthesis of T4 DNA ligase.

Applications

Cloning of restriction fragments, joining linkers and adapters to blunt-ended DNA, gene (gene fragments) synthesis.

Cohesive End Ligation

For most cohesive-end ligations, a 30 minute incubation at 20°C is sufficient. Incubations at 16°C for 4-16 hours are routinely used for the majority of applications.
Ligation of blunt-ends and single-base pair overhang fragments requires more enzyme to achieve the same extent of ligation as cohesive-end DNA fragments. Ligation can be enhanced by addition of PEG or by reducing the rATP concentration.

ATP is an essential cofactor for the reaction.

Storage buffer

• 50 mM KCl
• 10 mM Tris-HCl (pH 7.4)
• 0.1 mM EDTA
• 1 mM DTT
• 50% glycerol.

Unit definition

One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of lambda DNA in 30 minutes at 16°C at 5’ termini concentration of 0.12 µM (300 µg/ml). One Cohesive-End Ligation Unit equals 0.015 Weiss units. One Weiss unit equals 67 Cohesive-End Ligation Units.

Reaction buffer (10x)

• 50 mM Tris HCL (pH 7.8)
• 10 mM MgCl ₂
• 10 mM DTT
• 1 mM ATP.

Quality Assurance

Each lot of T4 DNA ligase is tested for endonucleases/exonucleases, in a blue/white cloning assay.

Heat inactivation

65 °C for 15 minutes or boiling for 2 minutes

Storage conditions

-20 °C