DNA Ligase (E.coli)

DNA Ligase (E.coli) catalyzes the formation of a phosphodiester bond between juxtaposed 5’ phosphate and 3’ hydroxyl cohesive termini in duplex DNA.

Description:

• Catalyzes the formation of a phosphodiester bond between duplex DNA fragments with cohesive ends.
• Condensation of the 5'-phosphoryl group with an adjacent 3'-hydroxyl group is coupled with the hydrolysis of NAD+.
• Suitable for high-efficiency cloning of full-length cDNA (1, 2).
  

  Unit Definition: One unit is defined as the amount of enzyme required to yield 50% ligation of Hind III fragments of lambda DNA. Incubation is at 16°C in 20 µl of assay mixture with a DNA terminus concentration of 0.02 µM (50 µg/ml).

  Storage Conditions: Store at –20°C.

  1 x Reaction Buffer: 30 mM Tris-HCl (pH 8.0 at 22°C), 1 mM dithiothreitol, 4 mM MgCl2, 26 µM NAD+, 50 µg/ml bovine serum albumin.

  Optimal ligation occurs at 16°C.

  Storage Buffer: 10 mM Tris-HCl (pH 7.4 at 22°C), 50 mM KCl, 0.1 mM EDTA, 10 mM ammonium sulfate, 1 mM dithiothreitol and 50% (v/v) glycerol.

  Quality Control: All preparations are tested for contaminating endonuclease and exonuclease activities, along with functional testing in the ligation reaction.

  Ligation Assay Conditions: 30 mM Tris-HCl (pH 8.0 at 22°C), 4 mM MgCl2, 1.2 mM EDTA, 1 mM dithiothreitol, 0.026 mM NAD+, 50 µg/ml bovine serum albumin and Hind III fragments of lambda DNA. Incubation is at 16°C for 30 min.

  References:

• Okayama, H. and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170.
• Gubler, U and Hoffman, B.J. (1983) Gene 25, 263-269.