Intact Genomics T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5’-phosphate and 3’-hydroxyl termini in duplex DNA or RNA. This enzymes joins DNA fragments with either cohesive or blunt termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1).
• Cloning of restriction enzyme generated DNA fragments
• Cloning of PCR products
• Next-gen library preparation
• Joining linkers and adapters to cohesive or blunt-ended DNA
• Nick repair in duplex DNA, RNA or DNA/RNA hybrids
• Self-circularization of linear DNA
The physical purity of Intact Genomics T4 DNA Ligase is ≥99% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below).
Intact Genomics T4 DNA Ligase displays significantly higher ligation efficiency than the nearest competitor.
E. coli strain expressing a recombinant clone
- T4 DNA Ligase
- 10x T4 DNA Ligase Reaction Buffer
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 ºC
1x T4 DNA Ligase Reaction Buffer
50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, pH 7.5 @ 25 ºC
One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (250 ng/µl) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA ligase reaction buffer.
Inhibition and Inactivation
- Inhibitors: metal chelators, phosphate and ammonium ions, KCl and NaCl at a concentration higher than 50 mM.
- Inactivated by heating at 70 °C for 15 min or by addition of EDTA.
Quality Control Assays
- Endonuclease Activity (Nicking)
1 µg of supercoiled plasmid DNA is incubated with 20 units of T4 DNA Ligase in 1x Ligase buffer for 2 hours at 37 ºC. Following incubation, the supercoiled DNA is visualized on an ethidium bromide stained agarose gel. No visible nicking or cutting of DNA was found.
- Functional Assay
DNA Ligase functional efficiency is tested in cloning assays.3210 3212 3216 3217