T4 DNA Ligase is a 55.3kDA monomeric enzyme that catalyzes the formation of phosphodiester bonds between adjacent 5´- phosphate and 3´-hydroxyl termini in double-stranded DNA and/or double-stranded RNA. T4 DNA Ligase is capable of joining blunt and complementary cohesive ends, making it the Ligase of choice for restriction-ligation cloning of DNA fragments into plasmids. Moreover, this enzyme can be used for nick-repair as it closes nicks in double-stranded DNA or DNA/RNA hybrids.
Source: Purified from E.coli harbouring gene 30 from bacteriophage T4
Quantity: 1000U at a concentration of 5 Weiss Units/µl in storage buffer (20mM Tris-HCl (pH 7.5), 50mM KCl, 1mM DTT, 0.1mM EDTA, and 50% glycerol), supplied with 1,5ml of T4 DNA Ligation Buffer (5X) containing ATP.
Unit Definition: One Weiss unit is defined as the amount of enzyme required to catalyze the conversion of 1nmol of [32PPi] into Norit®-absorbable form at 37ºC in 20 min, using specific reaction conditions (1). One Weiss units is the equivalent of approximately 200 CE units (Cohesive End ligation units), whereas one CEU is defined as the amount of enzyme required for 50% ligation of HindIII fragments of 1µg of lambda DNA at 16ºC in 30 min.
Properties: T4 DNA Ligase has an optimal reaction temperature of 22ºC but can be used in a wide temperature range (16ºC-37ºC). The enzyme can be heat inactivated at 65ºC for 10min, or chemically inactivated by NaCl or KCl at final concentrations above 200mM.
QC: Enzyme activity is assayed by incubation of 3.3µM [32PPi] in 66mM Tris-HCl (pH 7.6), 6.6mM MgCl2, 0.066mM ATP, 10mM DTT, as well as by a blue/white colony assay. Absence of endonucleases and exonucleases is ascertained by appropriate assays.
Storage: -20ºC for at least 1 year.