Tth DNA Ligase (250 U)

A thermostable ligase for ligase chain and detection reactions, offered by QIAGEN. The enzyme 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, RNA, or blunt-ended DNA.

The ligase is isolated from an Escherichia coli strain containing a plasmid carrying the Thermus thermophilus DNA ligase gene. It is supplied in a storage buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 1 mM DTT, and 50% glycerol. The accompanying 10x Tth ligation buffer contains 200 mM Tris-HCl (pH 8.3), 250 mM KCl, 100 mM MgCl2, 5 mM NAD, and 0.1% Triton X-100.

One unit of the ligase catalyzes the ligation of 50% of the cos sites present in 1 μg of bacteriophage lambda DNA in 1 minute at 45°C, with one unit equivalent to 15 cohesive end units (CEU). The product is supplied at a concentration of 5 U/μl (75 CEU/μl). The enzyme remains stable and active across 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 temperature. Quality control is performed in a reaction containing 1 μg of bacteriophage lambda DNA digested with SalI and SmaI in 1x Tth Ligation Buffer for 100 minutes at 45°C, with results assayed by agarose gel electrophoresis. Testing confirms the product is free of detectable DNase, exonuclease, endonuclease, and RNase contamination. Components are shipped on dry or blue ice and should be stored at -20°C; the product is intended for research use only.