Properties & Usage
Unit Definition
(Cohesive End Unit) One unit is defined as the amount of enzyme required to give 50% ligation of the 12-base pair cohesive ends of 1 µg of BstEII-digested λ DNA in a total reaction volume of 50 µl in 15 minutes at 45°C. A cohesive end unit is equivalent to the nick-closing unit (1). Reaction Conditions
1X 9°N DNA Ligase Reaction Buffer
Incubate at 45°C
1X 9°N DNA Ligase Reaction Buffer
10 mM Tris-HCl
600 µM ATP
2.5 mM DTT
2.5 mM MgCl2
0.1% Triton® X-100
(pH 7.5 @ 25°C)
Usage Concentration
40,000 units/ml
Storage Buffer
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
200 µg/ml Recombinant Albumin
50% Glycerol
10 mM ammonium sulfate
pH 7.4 @ 25°C
Heat Inactivation
No Unit Assay Conditions
1X 9°N DNA Ligase Reaction Buffer and 20 µg/ml BstEII-digested λ DNA in a 50 µl reaction. After incubation at 45°C for 15 minutes, the reaction is terminated by addition of stop dye (50% glycerol, 50 mM EDTA and bromophenol blue), heated at 70°C for 10 minutes and then loaded on a 0.7% agarose gel. Due to the presence of ligase, the cos ends of BstEII-digested λ DNA will stay together after 70°C heat treatment.