Description
Tth DNA Ligase catalyses the ligation between double-stranded DNA molecules with 3’ hydroxyl and 5’ phosphate termini. The enzyme will also repair single-stranded nicks in double-stranded nucleic acids.
Source
Isolated from an E. coli strain containing a plasmid carrying the Thermus thermophilus DNA ligase gene.
Features
Ligates double-stranded DNA
Repairs single strand nicks in double-stranded DNA
Concentration
50 units/µl
Applications
Tth DNA ligase is recommended for the ligase chain reaction (LCR).
Unit Definition
One nick-closing unit of the enzyme is defined as the amount of ligase that converts 0.5µg of nicked pBR322 to circular form in 15 minutes at 65°C under the following assay conditions: 20mM Tris-HCl (pH8.3 at 25°C), 50mM KCl, 10mM MgCl2, 1mM EDTA, 1mM NAD+, 10mM DTT, 0.5µg nicked pBR322, 0.1% (v/v) Triton® X-100.
Note
- Tth ligase has a half-life of 1 hour at 95 °C. The optimum temperature for nick-closing is 65–72 °C. In the presence of polyamines, Tth ligase is capable of ligating blunt-ended fragments. The optimum temperature for this is 65 °C.
- Tth DNA ligase comes with a 5 x reaction buffer containing 100 mM Tris-HCl (pH 8.3), 250 mM KCl, 50 mM MgCl2, 5 mM EDTA, 54 mM NAD+, 50 mM DTT, 0.5 % (v/v) Triton X-100