E.coli DNA ligase

Description:
E. coli DNA Ligase is an NAD+-dependent enzyme that catalyzes the formation of phosphodiester bonds between complementary 3´-hydroxyl and 5´-phosphoryl termini of dsDNA. The enzyme works best with cohesive dsDNA ends and is also active on nicked DNA. Blunt ends can be ligated in the presence of condensing reagents such as polyethylene glycol or Ficoll®. A 10X Reaction Buffer is provided with the enzyme.

Source:
E. coli strain containing an overproducing clone of E. coli DNA Ligase.

Specific Activity:
6000U/mg

Applications:

• Ligation for cloning.Primer Extension(delete)
• Okayama and Berg cDNA cloning

Unit Definition:
One unit of E. coli DNA Ligase is defined as the amount of enzyme required to provide ligation (>50%) of Hind III digested phage lambda DNA (5´ DNA termini concentration of 0.12 µM, 300 µg/ml) in 30 minutes at 16°C under standard assay conditions.

Reaction Conditions:
1X E. coli DNA Ligase Reaction Buffer
Incubate at 16°C

10X E. coli DNA Ligase Reaction Buffer
30 mM Tris-HCl
4 mM MgCl2
26 µM NAD
1 mM Dithiothreitol
50 µg/ml BSA
pH 8.0 @ 25°C

Recommended Storage Condition:
 -20°C

References:
1. Zimmerman, S. B. and Pheifer, B. H. (1983) Proc. Natl. Acad. Sci., USA 80, 5852-5856.
2. Okayama, H. and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170.