Tfi DNA Ligase

Tfi DNA Ligase는 DNA Ligase 의 일종으로서 절단된 이중나선 DNA분자의 인접한 5'-인산기 말단과 3'-수산기 말단사이를 phosphodiester bond로 연결해주는 효소입니다. 특히 반응 온도가 45℃와  65℃ 사이이므로 다른 T4 DNA Ligase, E. coli DNA ligase 등에 비해 더 높은 온도에서 활성이 안정하게 유지되어  높은 온도 조건에서 반응할 수 있습니다.

₩75,000
카탈로그 번호
E-3111-CFG

Tfi DNA Ligase는 DNA Ligase 의 일종으로서 절단된 이중나선 DNA분자의 인접한 5'-인산기 말단과 3'-수산기 말단사이를 phosphodiester bond로 연결해주는 효소입니다.
특히 반응 온도가 45℃와 65℃ 사이 이므로 다른 T4 DNA Ligase, E. coli DNA ligase 등에 비해 더 높은 온도에서 활성이 안정하게 유지되어  높은 온도조건에서도 반응할 수 있습니다.




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 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℃.

Concentration:

20 units/㎕

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℃.

Activity Assay Conditions

The activity assay is carried out in a 20 ㎕ reaction containing 1 ug of PspEI digested lambda DNA and 1 x Tfi DNA ligase reaction buffer. After incubation at 45℃ 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℃ 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.

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℃~ 65℃)

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℃, 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



Fig 2. Heat Stability test at 95℃

Incubate the enzyme at 95℃ each time. And then add 1 unit ligase to a 20 ㎕ reaction containing 1ug DNA[lambda PspEI] 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



Fig 3. Heat Stability test at 65℃

Incubate the enzyme for each time at 65℃ and then add 1 unit ligase to a 20 ㎕ reaction containing 1 ug DNA[lambda PspEI] 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

Manual

• Manual_Tfi DNA Ligase


MSDS

• MSDS_Tfi 10X Reaction buffer

• MSDS_Tfi DNA Ligase


Quality Assurance

Bioneer is the holder of Quality Management System Certificates for the following standards.

• ISO 9001 – certificate

• EN ISO 13485 - certificate


Cat. No. Product Description Price Add to Cart
E-3111 Tfi DNA Ligase, 2,000 U ₩75,000 add cart
E-3112 Tfi DNA Ligase, 10,000 U(5 x 2,000 U) ₩300,000 add cart












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