DNA Ligase, E coli

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Reagents Supplied

10X E coli DNA Ligase Reaction Buffer

Source

Escherichia coli

Applications

• Catalyzes the formation of a phosphodiester bond between duplex DNA fragments with cohesive ends
• Condensation of the 5'-phosphoryl group with 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 the amount of enzyme required to yield 50% ligation of HindIII fragments of lambda DNA in 30 min at 16°C in a 20 µl assay mixture containing a DNA terminus concentration of 0.12 µM (300µg/ml)

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
Hind III fragments of lambda DNA
Incubation at 16ºC for 30 minutes
Note: Optimal ligation occurs at 16°C

Reaction Buffer

30 mM Tris-HCL (pH 8.0 at 22°C)
1 mM dithiothreitol
4 mM MgCl₂
26 µM NAD+
1.2 mM EDTA

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 
50% (v/v) glycerol

Quality Control

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

Storage Conditions

Store at -20°C
Product shipped on dry ice

References

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