T4 DNA Ligase catalyzes the formation of a phosphodiester bond between the terminal 5′ phosphate and a 3′ hydroxyl groups of duplex DNA or RNA. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids. Cohesive ligation with T4 DNA Ligase can be completed in less than 10 min.
- Cloning of restriction fragments
- Joining linkers and adapters to blunt-ended DNA
- Library construction
- TA cloning
- Recirculization of linear DNA
Purified from a recombinant E. coli strain carrying the T4 DNA Ligase gene
- T4 DNA Ligase, 2,000,000 U/ml in storage buffer: 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol, pH 7.5 @ 25°C.
- 4 X T4 DNA Ligation Buffer: 250 mM Tris-HCI, 40 mM MgCl2, 4 mM DTT, 4 mM ATP, 30% PEG 6000, pH 7.6 @ 25°C
1 unit is defined as the amount of T4 DNA Ligase required to join 50% of 100 ng of DNA fragments with cohesive termini in 50 µl 1X T4 DNA Ligase Buffer following a 30 minute incubation at 23°C.
- The absence of endo-, exodeoxyribonucleases, phosphatases, and ribonucleases confirmed by appropriate quality tests.
- Functionally tested for the capacity to join cohesive- and blunt-end DNA fragments.