核糖核酸酶T1是一种来源于米曲霉 (Aspergillus oryzae) 的核糖核酸内切酶，可特异性地在单链 RNA 的鸟嘌呤核糖核苷酸 (G) 后进行切割，产生 3' 磷酸末端。 核糖核酸酶T1 能够形成核苷 2'，3-环磷酸中间体，以切割 3'-鸟苷残基与邻近核苷 5'-OH 基团之间的磷酸二酯键产生含末端 3'-GMP 的寡核苷酸和 3'-GMP。
核糖核酸酶T1 的活性不依赖于金属离子。
组分表
| R1522664 | 组件 | 物理外观 | 100KU | 5×100KU | 储存 | | R1522664A | 核糖核酸酶T1 (500 U/μl) | Liquid | 200ul | 5×200µl | -20℃. Avoid freeze/thaw cycle. | | R1522664B | 10× 核糖核酸酶T1缓冲液 | Liquid | 1ml | 5×1ml | -20℃. Avoid freeze/thaw cycle. |
|
活性定义
1 活性单位 (U) 是指在 37℃，pH7.5 条件下，以酵母 RNA 为底物， 使 A260 变化 1.0 所需要的酶量。
适用范围
1. 去除 DNA 中的 RNA；
2. 去除重组蛋白中的 RNA；
3. RNA 测序；
4. 用于核糖核酸酶保护试验（与 RNase A 结合使用）；
5. 测定不含 G 或低 G 含量 DNA 模板的转录水平。
质量控制
蛋白纯度
经 SDS-PAGE 凝胶电泳检测，蛋白纯度不低于 95%。
非特异性内切酶活性
37℃下，在 20 μl 反应体系中将 500 U 核糖核酸酶T1 与 200 ng 超螺旋质粒 DNA 共同温育 4 h，使用琼脂糖凝胶电泳检测，少于 20% 的质粒 DNA 转变成缺刻或线性状态。
DNase 活性
37℃下，在 20 μl 反应体系中将 500 U 核糖核酸酶T1 与 15 ng 双链 DNA 片段共同温育 16 h，使用琼脂糖凝胶电泳检测，双链 DNA 片段无变化。
使用方法
1. 反应体系
| 组分 | 体积 | 终浓度 |
|---|
| 单链 RNA（ssRNA） | 1~10 μg | 50~500 ng/μL | | 10× 核糖核酸酶T1缓冲液 | 2 μL | 1× | | RNase 抑制剂（40 U/μL）* | 1 μL | 2 U/μL | 核糖核酸酶T1 (500 U/μl)
| 0.4 μL | 10 U/μL | | 无核酸酶水 | 补至 20 μL | - |
|
注 : 为避免 RNase A 等环境中的 RNase 污染使底物 RNA 降解，建议在反应体系中加入RNase 抑制剂（货号：R350859），底物 RNA 应在 RNase 抑制剂加入并混匀之后加入。
2. 推荐的反应条件 37℃孵育 15 min。可根据实际情况适当延长或缩短孵育时间以达到最佳酶切效果。
注意事项
- 多种金属离子可抑制核糖核酸酶T1的活性，包括 Mg²⁺ (100 mM MgCl₂ 约抑制 40% 的活性)、Ca²⁺ (10 mM CaCl₂ 约抑制 30% 的活性 )、Zn²⁺、Fe²⁺、Cu²⁺ 等。除此之外，Guanylyl 2′-5′ guanosine 是核糖核酸酶T1的特异性抑制剂。
- 核糖核酸酶T1的热失活是可逆的，建议通过柱纯化或酚 / 氯仿抽提的方法去除核糖核酸酶T1。
- 在 pH 6.0 的溶液中， 核糖核酸酶T1可耐受 100℃加热 10 min；但在 pH > 9.0 的碱性溶液中不稳定。
- 为了您的安全和健康，请穿实验服并戴一次性手套及口罩进行实验操作。
RNase T1 is an endoribonuclease derived from Aspergillus oryzae. It specifically cleaves single-stranded RNA after guanosine residues (G), generating 3'-phosphate termini. RNase T1 forms nucleoside 2',3'-cyclic phosphate intermediates, cleaving the phosphodiester bond between the 3'-guanosine residue and the adjacent nucleoside 5'-OH group to produce oligonucleotides terminating in 3'-GMP and 3'-GMP.
The activity of RNase T1 is independent of metal ions.
Components List
R1522664
| Components | Appearance | 2KU | 5×2KU | Storage | | R1522664A | RNase T1 (500 U/μl) | Liquid | 200ul | 5×200µl | -20℃. Avoid freeze/thaw cycle. | | R1522664B | 10× RNase T1 Buffer | Liquid | 1ml | 5×1ml | -20℃. Avoid freeze/thaw cycle. |
|
Unit Definition
One unit (U) is defined as the amount of enzyme required to produce an absorbance change of 1.0 at A260 using yeast RNA as the substrate at 37 °C and pH 7.5.
Applications
- Removal of RNA contaminant from DNA preparations.
- Removal of RNA contaminant from recombinant protein preparations.
- RNA sequencing.
- Use in ribonuclease protection assays (in combination with RNase A).
- Quantitation of transcription levels from DNA templates with no or low guanine content.
Quality Control
Protein Purity
SDS-PAGE analysis confirms protein purity of no less than 95%.
Non-specific Endonuclease Activity
500 U of RNase T1 was incubated with 200 ng supercoiled plasmid DNA in a 20 μL reaction system at 37 °C for 4 hours. Agarose gel electrophoresis showed less than 20% of the plasmid DNA was converted to nicked or linear form.
DNase Activity
500 U of RNase T1 was incubated with 15 ng double-stranded DNA fragments in a 20 μL reaction system at 37 °C for 16 hours. Agarose gel electrophoresis showed no degradation of the dsDNA fragments.
Usage Protocol
1. Reaction system
| Component | Volume | Final Concentration |
|---|
| ssRNA | 1~10 μg | 50~500 ng/μL | | 10× RNase T1 Buffer | 2 μL | 1× | | RNase Inhibitor (40 U/μL)* | 1 μL | 2 U/μL | | RNase T1 (500 U/μL) | 0.4 μL | 10 U/μL | | Nuclease-Free Water | up to 20 μL | - |
|
Note: To prevent degradation of the substrate RNA caused by contamination with environmental RNases such as RNase A, it is recommended to add RNase Inhibitor (Cat.No.: R350859) to the reaction system. The substrate RNA should be added only after the RNase Inhibitor has been added and thoroughly mixed.
2. Recommended reaction conditions: Incubate at 37 °C for 15 minutes. The incubation time can be appropriately extended or shortened according to actual conditions to achieve optimal digestion results.
Notes
- The activity of RNase T1 can be inhibited by various metal ions, including Mg²⁺ (100 mM MgCl₂ inhibits approximately 40% of activity), Ca²⁺ (10 mM CaCl₂ inhibits approximately 30% of activity), Zn²⁺, Fe²⁺, Cu²⁺, etc. In addition, Guanylyl 2′-5′ guanosine is a specific inhibitor of RNase T1.
- Heat inactivation of RNase T1 is reversible. It is recommended to remove RNase T1 by column purification or phenol/chloroform extraction.
- RNase T1 is stable when heated at 100℃ for 10 min in solutions at pH 6.0, but unstable in alkaline solutions at pH > 9.0.
- For your safety and health, please wear a lab coat, disposable gloves, and a face mask during the experiment.
ZJ26F0636886
危险品化学品经营许可证（带存储）