E. coli DNA Ligase (2500 U)

From QIAGEN's assay technologies portfolio, this recombinant E. coli DNA ligase catalyzes the formation of phosphodiester bonds between an adjacent 5ʹ phosphate and a 3ʹ hydroxyl of DNA ends, requiring NAD+ and Mg2+ as cofactors. It is suited to molecular biology applications such as cDNA cloning, and blunt-ended DNA ligation is extremely inefficient relative to cohesive DNA end ligation and nick sealing.

The recombinant protein is expressed from a plasmid encoding E. coli DNA Ligase in E. coli and has a molecular weight of 73.6 KDa. One unit is defined as the amount of enzyme required to ligate 50% of 100 ng DNA fragments with cohesive termini in 30 minutes at 25°C. The enzyme is supplied at a concentration of 10,000 U/mL in 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, and 50% glycerol (pH 7.5 at 25°C), accompanied by a 10X reaction buffer containing 300 mM Tris-HCl, 40 mM MgCl2, 10 mM DTT, 260 µM NAD, and 0.5 mg/mL BSA (pH 8.0 at 25°C). Storage temperature is -25°C to -15°C.

Quality control specifications include a purity of >99%, a specific activity of 20,000–28,880 U/mg, <2.0% single-stranded exonuclease release (100 U tested), <1.0% double-stranded exonuclease release (100 U tested), no double-stranded endonuclease conversion (100 U tested), and <10 copies of E. coli DNA contamination (50 U tested). Protein concentration is determined by OD280 absorbance and physical purity is evaluated by SDS-PAGE with silver-stain detection. The product was developed, manufactured, and sold for in vitro use only and is not suitable for administration to humans or animals.