Description
Tfi DNA Ligase is a thermostable enzyme isolated form Thermus filiformis. It catalyzes the formation of phosphodiester bond between double stranded DNAs or between oligonucleotide 3’-hydroxyl end and 5’-phosphate end.
The enzyme is active at high temperatures between 45°C and 65°C what makes it special among other ligases like T4 DNA Ligase or E.coli DNA Ligase.
Tfi DNA ligase is an enzyme of choice for all ligations to be carried at high temperatures. It is used for such applications as Ligase Chain Reaction (LCR), Oligonucleotide Ligation Assay (OLA) or mutagenesis by incorporation of a phosphorylated oligo during PCR.
Specifications
Applications
- Ligase Chain Reaction (LCR)
- Oligonucleotide Ligation Assay (OLA)
- Mutagenesis by Incorporation of a phosphorylated oligo during PCR Amplification
- Simultaneous Mutagenesis of Multiple Sites
Supplied Components
10 x Reaction Buffer (1 ml): 300 mM Tris-HCl (pH8.3), 250 mM KCl, 50 mM MgCl2, 5 mM NAD
1 x Dilution Buffer (1 ml): 10 mM Tris-HCl (pH7.6), 0.1 mM EDTA, 50 mM KCl, 1 mM DTT, 200 ug/mL acetylated BSA, 50% Glycerol
Storage Conditions
20 mM Tris-HCl (pH7.6), 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.5% Tween -20, 0.5% IGEPAL CA-630, 50% Glycerol, store at -20°C.
Concentration
20 units/μl
Unit definition
One unit of Tfi DNA Ligase is defined as the amount of enzyme required to give 50% ligation of the 12 base pair cohesive ends of 1 ug of PspEI digested lambda DNA in 10min at 45°C.
Activity Assay Conditions
The activity assay is carried out in a 20 μl reaction containing 1 ug of PspEI digested lambda DNA and 1 x Tfi DNA ligase reaction buffer. After incubation at 45°C for 10 min., the reaction is terminated by addition of stop solution (40%(w/v) sucrose, 50 mM EDTA and 0.25% bromophenol blue). Then heat at 70°C for 10 min and immediately load on a 0.8% agarose gel.
Stability
The half-life of the enzyme in 1 x reaction buffer is more than 1 hour at 95°C and 55 hours at 65°C.
Note
Tfi DNA Ligase should not be used as a substitute for other DNA ligase, i.e., T4 DNA Ligase.
Supporting Data
Manual
References
Resources
Manual
MSDS
Quality Assurance
Bioneer is the holder of Quality Management System Certificates for the following standards.
