Description
Tth DNA Ligase catalyzes the NAD-dependent formation of phosphodiester bonds between adjacent 3’-hydroxyl and 5’-phosphate termini in double-stranded DNA. It is not active against single-stranded DNA or RNA and blunt-ended DNA. The enzyme is isolated from the Escherichia coli strain containing a plasmid carrying the Thermus thermophilus DNA ligase gene. It is supplied with 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 1 mM DTT, 50% glycerol. One unit of Tth DNA Ligase catalyzes the ligation of 50% of the cos sites present in 1 μg of bacteriophage lambda DNA in 1 minute at 45°C. One unit of Tth DNA Ligaseiq equivalent to 15 cohesive end units (CEU).
Assay Specification Ligase activity Passed DNase contamination None detected Exonuclease activity None detected Endonuclease activity None detected
Tth DNA Ligase is stable and active in an optimum ligation temperature range of 45–65°C, which is 7–10°C higher than T4 DNA ligase. The Tm of the substrates determines the final reaction ligation temperature. High ligation temperature eliminates nonspecific ligation.
Quality Control Tth DNA ligase activity is assayed in a reaction containing 1 µg of bacteriophage lambda DNA digested with SalI and SmaI (Control DNA), 1x Tth Ligation Buffer and varying amounts of enzyme for 100 minutes at 450°C. Results are assayed by agarose gel electrophoresis. The product is free of unspecific DNA nucleases. Results are assayed by agarose gel electrophoresis following incubation of 1 µg of DNA substrate with 5 U of Tth ligase enzyme for 4 hours at 700°C.
