T4 DNA Ligase

$ 96.14

Category: Accessories

Description 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 enzyme joins DNA fragments with either cohesive or blunt termini and repairs single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1). Applications • 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 Purity The physical purity of Intact Genomics T4 DNA Ligase is ≥99% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below). Comparison Testing Intact Genomics T4 DNA displays significantly higher ligation efficiency than the nearest competitor. Product Source E. coli strain expressing a recombinant clone Product Includes T4 DNA Ligase 10x T4 DNA Ligase Reaction Buffer Storage 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 Reaction Buffer 50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, pH 7.5 @ 25 ºC Unit Definition 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.3212 3216 3217

T4 DNA Ligase

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