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.
T4 DNA Ligase
|402002||2000 U||Add to cart|
T4 DNA Ligase
|402010||10000 U||Add to cart|
50 – 100 units/μl
Purified from E. coli strain harbouring the plasmid that directs the synthesis of T4 DNA ligase.
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.
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)
Each lot of T4 DNA ligase is tested for endonucleases/exonucleases, in a blue/white cloning assay.
65 °C for 15 minutes or boiling for 2 minutes