|T4 DNA Ligase catalyzes formation 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 duplexDNA, RNA or DNA/RNA hybrids.Purified from E. coli.|
|A solution of 50 – 100 units/μl in a buffer containing:
50 mM KCl
|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:
50 mM Tris HCL (pH 7.5)
|Absence of endonucleases/exonucleases activity|
|Cloning of restriction fragments, joining linkers and adapters to blunt-ended DNA, gene (gene fragments) synthesis|
|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.
|Heating at 65 °C for 15 minutes or boiling for 2 minutes|
|Store at -20°C. Can be shipped on ice packs.|
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