Enzyme Properties
Heat Inactivation: 70°C for 10 minutes
Application
Blunt or cohesive-end ligation
Repair of nicks in double-stranded nucleic acids
Reagents Supplied
10 x Reaction Buffer: 500 mM Tris-HCI (pH 7.8), 100 mM MgCl2, 50 mM DTT, 10 mM ATP, 25 μg/ml BSA
Concentration
200 U/μl
Storage Conditions
50% glycerol containing 10 mM Tris-HCI (pH 7.5), 50 mM KCI, 1 mM EDTA, 10 mM 2-Mercaptoethanol
Store Temperature
-20°C
Unit Definition
0.01 Weiss unit of enzyme is defined as the amount of enzyme required to give 90% ligation of Hind III fragments of lambda DNA in 30 minutes, at 16°C in 20 μl of the assay mixture.
Lane 1: DNA fragment (digested with EcoR V)
Lane 2, 3, 4, 5: T4 DNA Ligase 1 U, 16°C, 10, 20, 30 and 60 minutes
Lane 6, 7, 8, 9: T4 DNA Ligase 1 U, 25°C, 10, 20, 30 and 60 minutes
Lane 10, 11, 12,13: T4 DNA Ligase 1 U, 37°C, 10, 20, 30 and 60 minutes
Lane 14: Lambda DNA (digested with Hind III)
Lane 15, 16, 17: T4 DNA Ligase 1 U, 16°C, 10, 20 and 30 minutes
Lane 18, 19, 20: T4 DNA Ligase 1 U, 25°C, 10, 20 and 30 minutes
Lane 21, 22, 23: T4 DNA Ligase 1 U, 37°C, 10, 20 and 30 minutes