신제품 Thermostable USER (Uracil-Specific Excision Reagent) III Enzyme은 uracil base(U)의 절단을 촉매하여 single nucleotide gap을 만들어 주는 효소입니다. 이 효소는 50-75°C (optimal activity observed at 65°C)에서 활성화 되며, 열로 비활성화 시킬 수 없습니다. Column purification 또는 phenol chloroform extraction 방법으로 제거해 줘야 합니다.
![[NEB] Thermostable USER® III Enzyme M5509](https://www.korambiotech.com/wp-content/uploads/2022/12/M5509.jpg)
Thermostable USER® III Enzyme
• Thermostable USER III Enzyme is a mixture of Afu Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease IV.
• Thermostable USER III Enzyme also has 3′-diesterase activity which can remove 3′ phosphate, 3′-α, β-unsaturated aldehyde, phosphoglycoaldehyde, and other 3′ blocking groups (Leaves 3′-OH and 5′-dRP).
Reaction Conditions:
Thermostable USER III Enzyme is active between 50-75°C.
1X ThermoPol® Reaction Buffer Pack
Incubate at 65°C
Figure 1: Thermostable USER III Enzyme
![[NEB] Thermostable USER® III Enzyme](https://www.korambiotech.com/wp-content/uploads/2022/12/캡처.jpg)
Afu Uracil DNA glycosylase (UDG) catalyzes the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease IV breaks the phosphodiester backbone at the 3′ and 5′ sides of the abasic site so that base-free deoxyribose is released.
Figure 2: USER Enzyme (NEB #M5505), Thermolabile USER II Enzyme (NEB #M5508) and Thermostable USER III Enzyme (NEB #M5509) generate different functional ends after cleavage of DNA.
![[NEB] Thermostable USER® III Enzyme](https://www.korambiotech.com/wp-content/uploads/2022/12/2.jpg)
In addition to different optimal reaction temperatures (37°C for USER and Thermolabile USER II Enzyme and 65°C for Thermostable USER III Enzyme) and ability to be heat inactivated (Thermolabile USER II Enzyme only), the different USER Enzymes generate different 3’ and 5’ termini after cleavage. USER Enzyme (NEB #M5505) contains Endonuclease VIII and leaves a 3’ and 5’ phosphate after cleavage. Thermolabile USER II Enzyme (NEB #M5508) contains Endonuclease III and leaves a 3′-phospho-α, β-unsaturated aldehyde and 5′ phosphate after cleavage. Thermostable USER III Enzyme (NEB #M5509) contains Endonuclease IV and leaves a 3′-hydroxyl and 5′-deoxyribose phosphate.
Q&A
Thermostable USER III Enzyme is 100% active in Thermopol Buffer and has limited activity in other buffers. Activity in NEBuffers is as follows:
| NEBuffer | r1.1 | r2.1 | r3.1 | rCutSmat |
| % Activity | 100 | 50 | 50 | 75 |
Additionally, Thermostable USER III Enzyme is active in some polymerase and PCR buffers (see chart below).
| Buffer | Activity (Relative to Thermopol Buffer) |
|---|---|
| Thermopol II Reaction Buffer (NEB #B9004) | 100% |
| Q5® Reaction Buffer (NEB #B9027) | 50% |
| Colorless GoTaq® Flexi Buffer (Promega®) | 75% |
| PCR Buffer (Qiagen®) | 75% |
| iTaq Buffer | 100% |
| AmpliTaq Gold® 360 Buffer (ThermoFisher®) | 100% |
| Fast Start™ Taq DNA Pol PCR Buffer (Sigma Aldrich) | 0% |
| Ex Taq Buffer (Takara) | 0% |
| Dream Taq Buffer (ThermoFisher®) | 75% |
| Platinum II PCR Buffer (Invitrogen) | 75% |
Thermostable USER III Enzyme is active at temperatures >50°C, with optimal activity observed at 65°C. Minimal activity is observed at temperatures less than 50°C. Like USER Enzyme (NEB #M5505), Thermostable USER III Enzyme cannot be heat inactivated and must be removed by other methods (e.g., column purification/phenol chloroform extraction).
Thermostable USER III Enzyme will remove uracil from ssDNA with the same efficiency as from dsDNA. Thermostable USER III Enzyme has no activity on nucleotides (dUTP) in solution.
DNA와 RNA의 mole을 mass로 전환, ligation량 결정, OD 값에 따른 농도 등 실험에 필요한 각종 계산을 쉽게 할 수 있는 Tool입니다.
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