本产品是重组表达来源的 DNase I，与野生型相比，本产品经过了蛋白质工程改造，获得了远优于野生型的耐盐性，在 300 mM 一价 盐的溶液中仍能完全消化 DNA。本品不含 RNase 污染。
组分表
D1522668
| 组件 | 物理外观 | 1KU | 储存 | | D1522668A | 耐盐脱氧核糖核酸酶I (2 U/μl)
| Liquid | 500μl | -20℃. Avoid freeze/thaw cycle. | | D1522668B | 10× DNase I-ST 缓冲液
| Liquid | 1.25ml | -20℃. Avoid freeze/thaw cycle. |
|
活性定义
1 活性单位 (U) 是指在 37℃ 10 min 能够完全降解 1μg pUC19 质粒 DNA 所需要的酶量。
适用范围
1. 体外转录：使用 RNA 聚合酶进行体外转录后，用于模板 DNA 的去除。
2. 去除 RNA 样品中的基因组 DNA 污染。
3. 用于 DNase I 足迹法 (DNase I footprinting) 分析 DNA- 蛋白质相互作用。
4. 与 DNA Polymerase I 配合使用，用于缺口平移法标记 DNA。
5. 用于 DNA 随机片段文库构建。
6. 细胞凋亡 TUNEL 检测中部分剪切基因组 DNA 作为阳性对照。
质量控制
蛋白质纯度
使用 SDS-PAGE 凝胶电泳检测，蛋白纯度不低于 90%。
RNase 残留检测
将 2 U 耐盐脱氧核糖核酸酶I 与 500 ng 总 RNA 在 37℃温育 1 h，使用琼脂糖凝胶电泳检测超过 90% 的 RNA 仍保持完整。
使用方法
1. 去除 RNA 样品中的基因组 DNA 污染
加样体系：
| 组分 | 体积 |
|---|
| RNA | 1 μg | | 10× DNase I-ST 缓冲液 | 1 μl | 耐盐脱氧核糖核酸酶I (2 U/μl)
| 0.5 μl | | 无核酸酶水 | 补至 10 μl |
|
反应条件：37℃ 15 min。
失活条件：柱纯化法或者苯酚 / 氯仿抽提。
2. 体外转录后模板
DNA 的去除
操作步骤如下：
① 每 1 μg 模板 DNA 的转录反应体系中加入 1 U 耐盐脱氧核糖核酸酶I，酶的用量可以根据实际需要优化。
② 反应条件：37℃ 15 min。
③ 失活条件：柱纯化法或者苯酚 / 氯仿抽提。
注意事项
1. 进行 RNA 样品操作时请在 RNase-free 管中进行加样。
2. 在使用本品进行 RNA 样品中 DNA 的去除实验时，可在反应体系中添加终浓度 1 U/μl RNase Inhibitor, Murine 以保护 RNA 不被降解。
3. 耐盐脱氧核糖核酸酶I 对物理变性敏感，混匀时请勿剧烈振荡。
4. 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。
This product is recombinant DNase I. Compared with the wild-type enzyme, it has been engineered to exhibit significantly enhanced salt tolerance, capable of completely digesting DNA in solutions containing 300 mM monovalent salt. This product is free of RNase contamination.
Components List
D1522668
| Components | Appearance
| 1KU | Storage |
|---|
| D1522668A | DNase I-ST (2 U/μl) | Liquid | 500μl | -20℃. Avoid freeze/thaw cycles. | | D1522668B | 10× DNase I-ST Buffer | Liquid | 1.25ml | -20℃. Avoid freeze/thaw cycles. |
|
Activity Definition
One unit (U) is defined as the amount of enzyme required to completely degrade 1 μg of pUC19 plasmid DNA in 10 minutes at 37℃.
Applications
In vitro transcription: Removal of template DNA after RNA polymerase-mediated in vitro transcription.
Removal of genomic DNA contamination from RNA samples.
DNase I footprinting for analyzing DNA-protein interactions.
Nick translation labeling of DNA in combination with DNA Polymerase I.
Construction of random DNA fragment libraries.
Partial digestion of genomic DNA as a positive control in the TUNEL assay for apoptosis detection.
Quality Control
Protein Purity
Determined by SDS-PAGE, the protein purity is ≥90%.
RNase Residue Detection
2 U of DNase I-ST is incubated with 500 ng of total RNA at 37℃ for 1 h. More than 90% of the RNA remains intact as determined by agarose gel electrophoresis.
Protocol
1. Removal of Genomic DNA Contamination from RNA Samples
Reaction Setup:
| Component | Volume |
|---|
| RNA | 1 μg | | 10× DNase I-ST Buffer | 1 μl | | DNase I-ST (2 U/μl) | 0.5 μl | | Nuclease-Free Water | up to 10 μl |
|
Reaction Conditions: 37℃ for 15 min.
Inactivation: Column purification or phenol/chloroform extraction.
2. Removal of Template DNA after In Vitro Transcription
Procedure:
① Add 1 U of DNase I-ST per 1 μg of template DNA in the transcription reaction. The enzyme dosage can be optimized as needed.
② Reaction Conditions: 37℃ for 15 min.
③ Inactivation: Column purification or phenol/chloroform extraction.
Notes
- Perform all operations on RNA samples in RNase-free tubes.
- When using this product to remove DNA from RNA samples, Murine RNase Inhibitor can be added to the reaction system at a final concentration of 1 U/μl to protect RNA from degradation.
- DNase I-ST is sensitive to physical denaturation. Do not vortex vigorously when mixing.
- For your safety and health, please wear a lab coat, disposable gloves, and a face mask during the experiment.
ZJ26F0636352
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