Thermus thermophilus DNA ligase (recombinant form of the enzyme; cloned from strain HB27) catalyses the formation of a phosphodiester bond between the 5`-phosphate and 3`-hydroxil groups of adjacent nucleotides which are hybridized to a complementary target DNA. The ligation will occur only if the oligonucleotides are perfectly paired to the complementary target DNA and have no gaps between them. There fore, a single-base substitution can be detected.
One unit is defined as the amount of the enzyme required to give 50% (cohesive end unit) ligation of the 12- base pair cohesive ends of 1 µg of BstE II-digested ë-DNA in 15 minutes at 45°C in a total reaction volume of 50 µl. One cohesive end ligation unit equals 0,015 Weiss units.
10 mM Tris-HCl pH 7.5; 50 mM KCl; 0.1 mM EDTA; 1 mM DTT; 0.2% Triton X-100; 50% glycerol
10X REACTION BUFER
200 mM Tris-HCl pH 7.6; 250 mM KCl; 100 mM MgCl2, 100 mM DTT; 10 mM NAD, 1% Triton X-100
Optimal ligation occurs at 45°C.
Activity, SDS-PAGE purity, absence of RNases, ssDNases, endonucleases and phosphatases.