Thermostable Thermus Filiformis (Tfi) DNA Ligase

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Thermostable Thermus Filiformis (Tfi) DNA Ligase

Tfi DNA Ligase plays role of formatting phosphodiester bond by connecting double stranded DNA or connecting oligonucleotides 3’-hydroxyl end and 5’-phosphate end together.
Especially the reaction temperatures are between 45°C and 65°C compared to different DNA Ligase such as T4 DNA Ligase, E.coli DNA Ligase, etc.
It is the higher temperature that keeps safe and active. In addition, Tfi DNA Ligase is possible to use in ligation reaction in high reaction temperature requirement.

Application

- Ligase Chain Reaction (LCR)
- Oligonucleotide Ligation Assay (OLA)
- Mutagenesis by Incorporation of a phosphorylated oligo during PCR Amplification
- Simultaneous Mutagenesis of Multiple Sites

Supplied with Enzyme

- 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 Condition

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.

References

1. Barany, F. (1991) Proc. Natl. Acad. Sci. USA, 88, 189 - 193.
2. Landegren, U. et al.(1988) Science 241, 1077 - 1080
3. Michael, Scott F. (1994) Biotechniques 16:3, 410 - 412.
4.Gerard J. A. et al. (1993) Biotechniques 15:1, 172 - 178.

Experimental Data

Fig 1. Ligation test at various temperatures (45°C~ 65°C)

Incubate the reaction containing ligase 1 unit and 1 ug DNA[lambda PspEI] at each temperature for 10 min.
Lane 1: λ DNA/PspE I (control) Lane 2: Incubate at 45°C, 10 min
Lane 3: Incubate at 50°C, 10 min Lane 4: Incubate at 55°C, 10 min
Lane 5: Incubate at 60°C, 10 min Lane 6: Incubate at 65°C, 10 min

Fig 2. Heat Stability test at 95°C

Incubate the enzyme at 95°C each time. And then add 1 unit ligase to a 20 ul reaction containing 1ug DNA[lambda PspEI] and incubate the mixture at 45°C for 10 min.
Lane 1: λ DNA/PspE I (control) Lane 2: Incubate at 95°C, 10 min
Lane 3: Incubate at 95°C, 20 min Lane 4: Incubate at 95°C, 30 min
Lane 5: Incubate at 95°C, 40 min Lane 6: Incubate at 95°C, 50 min
Lane 7: Incubate at 95°C, 60 min Lane 8: Incubate at 95°C, 70 min
Lane 9: Incubate at 95°C, 80 min Lane 9: Incubate at 95°C, 90 min

Fig 3. Heat Stability test at 65°C

Incubate the enzyme for each time at 65°C and then add 1 unit ligase to a 20 ul reaction containing 1 ug DNA[lambda PspEI] and incubate the mixture at 4°C for 10 min.
Lane 1: λ DNA/PspE I (control) Lane 2: Incubate at 65°C, 6 hrs
Lane 3: Incubate at 65°C, 12 hrs Lane 4: Incubate at 65°C, 18 hrs
Lane 5: Incubate at 65°C, 24 hrs Lane 6: Incubate at 65°C, 30 hrs
Lane 7: Incubate at 65°C, 36 hrs Lane 8: Incubate at 65°C, 42 hrs
Lane 9: Incubate at 65°C, 48 hrs Lane 10: Incubate at 65°C, 54 hrs
Lane 11: Incubate at 65°C, 60 hrs Lane 12: Incubate at 65°C, 66 hrs
Lane 13: Incubate at 65°C, 72 hrs Lane 14:Incubate at 65°C, 78 hrs