▶ 응용 및 적용
• Ligase Chain Reaction (LCR)
• Oligonucleotide Ligation Assay (OLA)
• Mutagenesis by Incorporation of a phosphorylated oligo during PCR Amplification
• Simultaneous Mutagenesis of Multiple Sites
▶ 제품 구성
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Component
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E-3111
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Tfi DNA Ligase(2,000 U)
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100 μl
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10X Reaction buffer
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1 ml
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Dilution buffer
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1 ml
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• 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
▶ 농도
2,000 units (20 U/μl)
▶ 저장 조건
▶ 보관 온도
-20℃
▶ 단위(Unit) 정의
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 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.
▶ 안정도
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.
▶ 실험 자료
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
