Tfi DNA Ligase (10,000 units)

Tfi DNA Ligase binds between 5'-phosphate terminal and the 3'-hydroxyl terminal of to the truncated double-stranded DNA molecules with a phosphodiester bond. As the reaction temperature between 45 ° C and 65 ° C, its activity can be kept especially stable even at higher temperature than other T4 DNA ligases and E. coli DNA ligases.

※This product is shipped in dry ice.

$660.00

Cat. No.

E-3112

Product Overview

Features and Benefits

Thermostability
High resistance to heat allow stable reactions than T4 DNA Ligase, E. coli DNA ligase
Reproducibility
Reproducible results with uniform quality products supplied for each batch under the ISO 9001 quality system

Experimental data

Figure 1. Ligation test at various temperatures (45℃ ~ 65℃)

Incubate the reaction containing ligase 1 unit and 1 ug DNA [lambda/PspE I] at each temperature for 10 min.

- Lane 1: λ DNA/PspE I (control)    - Lane 2: Incubate at 45℃, 10 min
- Lane 3: Incubate at 50℃, 10 min         - Lane 4: Incubate at 55℃, 10 min
- Lane 5: Incubate at 60℃, 10 min         - Lane 6: Incubate at 65℃, 10 min

Technical/Specs

▶ Application

• Ligase Chain Reaction (LCR)

• Oligonucleotide Ligation Assay (OLA)

• Mutagenesis by Incorporation of a phosphorylated oligo during PCR Amplification

• Simultaneous Mutagenesis of Multiple Sites

▶ Components

Component	E-3111
Tfi DNA Ligase(2,000 U)	100 μl
10X Reaction buffer	1 ml
Dilution buffer	1 ml

• 10 x Reaction Buffer : 300 mM Tris-HCl (pH8.3), 250 mM KCl, 50 mM MgCl₂, 5 mM NAD
• 1 x Dilution Buffer : 20 mM Tris-HCl (pH 7.6), 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, Stabilizers, 50% Glycerol

▶ Concentration

2,000 units (20 U/μl)

▶ Storage Conditions

20 mM Tris-HCl (pH 7.6), 2 mM MgCl₂, 1 mM EDTA, 1 mM DTT, Stabilizers, 50% Glycerol

▶ Storage Temperature

-20℃

▶ 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 PspE I digested lambda DNA in 10min at 45℃.

▶ Activity Assay Conditions

The activity assay is carried out in a 20 μl reaction containing 1 ug of Psp E I digested lambda DNA and 1 x Tfi DNA ligase reaction buffer. After incubation at 45℃ for 10 min, the reaction is terminated by an addition of a stop solution (40% (w/v) sucrose, 50 mM EDTA and 0.25% bromophenol blue). Then heat at 70℃ 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℃ and 55 hours at 65℃.
Note: Tfi DNA Ligase should not be used as a substitute for other DNA ligase, i.e., T4 DNA Ligase.

▶ Experimental data

Figure 1. Heat Stability test at 95℃

Incubate the enzyme at 95℃ each time. And then add 1 unit ligase to a 20 μl reaction containing 1ug DNA [lambda/PspE I] and incubate the mixture at 45℃ for 10 min.

- Lane 1: λ DNA/PspE I (control) - Lane 2: Incubate at 95℃, 10 min
- Lane 3: Incubate at 95℃, 20 min       - Lane 4: Incubate at 95℃, 30 min
- Lane 5: Incubate at 95℃, 40 min       - Lane 6: Incubate at 95℃, 50 min
- Lane 7: Incubate at 95℃, 60 min       - Lane 8: Incubate at 95℃, 70 min
- Lane 9: Incubate at 95℃, 80 min      - Lane 10: Incubate at 95℃, 90 min

Figure 2. Heat Stability test at 65℃

Incubate the enzyme for each time at 65℃ and then add 1 unit ligase to a 20 μl reaction containing 1 ug DNA [lambda/PspE I] and incubate the mixture at 45℃ for 10 min.

- Lane 1: λ DNA/PspE I (control)    - Lane 2: Incubate at 65℃, 6 hrs
- Lane 3: Incubate at 65℃, 12 hrs        - Lane 4: Incubate at 65℃, 18 hrs
- Lane 5: Incubate at 65℃, 24 hrs        - Lane 6: Incubate at 65℃, 30 hrs
- Lane 7: Incubate at 65℃, 36 hrs        - Lane 8: Incubate at 65℃, 42 hrs
- Lane 9: Incubate at 65℃, 48 hrs        - Lane 10: Incubate at 65℃, 54 hrs
- Lane 11: Incubate at 65℃, 60 hrs - Lane 12: Incubate at 65℃, 66 hrs
- Lane 13: Incubate at 65℃, 72 hrs - Lane 14:Incubate at 65℃, 78 hrs

Literature/Support