本公司生产的 Exonuclease I，即核酸外切酶 I，是一种大肠杆菌来源的，能够特异催化单链线性 DNA (Single-strand linear DNA)从 3'到 5'方向降解的核酸外切酶。Exonuclease I 对底物具有高度选择性，只能特异催化具有 3'-OH 末端的单链线性 DNA 沿 3'到 5'方向降解，同时逐步释放单个 dNMP，直至 5'端残留两个脱氧核苷酸；不能酶解 3'端被其它基团修饰封闭的单链 DNA，也不能水解双链 DNA 和 RNA。Exonuclease I 常用于在 PCR 反应后或测序反应前，消除体系中残留的过量单链 DNA 引物；但不能用于带有 3'粘性末端的双链 DNA 的末端平滑，Exonuclease I 主要用于单链 DNA 从 3'到 5'方向逐步切除单个核苷酸。
| 来源（Source） | 大肠杆菌重组表达 | | 外观（Appearance） | 无菌液体 | | 保存液（Storage Buffer） | 10mM Tris-HCl, 100mM NaCl, 5mM DTT, 0.5mM EDTA, 100µg/ml BSA, 50% Glycerol, pH7.5 @ 25ºC.
| | 酶浓度（Enzyme Concentration） | 20 U/μL | | 纯度（Purity） | 不含除 Exonuclease I 以外的其它种类 DNA/RNA 内切酶和外切酶，不含磷酸酯酶。
| | 活性定义（Activity Definition） | One unit is defined as the amount of enzyme that will catalyze the release of 10nmol of acid-soluble nucleotide in a total reaction volume of 50μl in 30 minutes at 37 º C in 1X Exonuclease I Reaction Buffer with 0.17mg/ml single-stranded [3H]-DNA. |
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组分表
| E666128 | 组件 | 物理外观 | 2KU | 5×2KU | 25×2KU | 储存 | | E666128A | Exonuclease I, 20 U/μL | Liquid | 100ul | 5×100µl | 25×100µl | -20℃. Avoid freeze/thaw cycle. | | E666128B | 10×Exo I Reaction Buffer | Liquid | 400µl | 5×400µl | 25×400µl | -20℃. Avoid freeze/thaw cycle. |
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产品应用
常用于 PCR 反应产物中单链引物的去除；在进行 Sanger DNA 测序或 SNP 分析之前，去除 PCR 反应体系中的单链引物；去除样品中的单链线性 DNA，保留双链 DNA；分析样品中 3'-羟基末端单链 DNA 。
产品优势
Exonuclease I 常用于在 PCR 反应后或测序反应前，消除体系中残留的过量单链 DNA 引物。
使用说明
1.配制反应体系。 用于 DNA 样品中单链 DNA 的消除时，可参考下表在冰浴中配制如下反应体系(以 20μl 体系为例)：
| 试剂 | 体积 | 终浓度 |
|---|
超纯水
| (17.5-X) µL | - | 10× 反应缓冲液
| 2 µL | 1X | | DNA 样本 | X µL (100 nmol bases) | 5 nmol/µL | | 核酸外切酶I (20 U/µL) | 0.5 µL | 0.5 U/µL | 总体积
| 20 µL | - |
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用于 PCR 反应产物中单链 DNA 的消除时，可参考下表在冰浴中配制如下反应体系(以 5μl 体系为例)：
| 试剂 | 体积 | 终浓度 |
|---|
| PCR 产物 | 4.5 µL | - | 核酸外切酶I (20 U/µL)
| 0.5 µL | 2 U/µL | | 总体积 | 5 µL | - |
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注 1：上述表格中 DNA Sample 的用量已经比较大，对于样品比较少的情况下，完全可以大幅减少 DNA 样品的加入量。
注 2：由于涉及 DNA 操作，需要严格按照 DNA 操作的规范进行，相关试剂和耗材须避免 DNase 污染，确保是 DNase free 的。
注 3：为获得最好的底物酶切效果，也可根据实际情况，参考酶活单位定义适当调整底物与酶的用量，摸索最优酶促反应体系。
注 4：如果同时进行多个连接反应，可以把上表中除底物之外的所有溶液和酶提前预混合均匀，然后分装到各反应管内，最后再加入底物。
注 5：Exonuclease I 在绝大多数 PCR 体系中也具有活性，可直接在 PCR 产物中加入适量 Exonuclease I 进行酶切反应，用于单链引物 的去除。
注 6：如果 Exonuclease I 在某种特殊的反应体系中对单链 DNA 的酶切效果较差，建议搭配 Exonuclease I 的 10X Reaction Buffer 进 行单链 DNA 的消除反应。
2.按上表配制好反应体系后，适当轻轻混匀反应体系，随后离心数秒使粘附在管壁上的液体沉至管底。
3.反应条件：37ºC 孵育 30 分钟。 注：反应时间可以根据实际情况酌情适当调节。
4.终止反应：80ºC 孵育 20 分钟可使 Exonuclease I 充分失活。
5.根据使用需要，酌情取消化后的产物进行聚丙烯凝胶电泳分析，拍照观察并分析效果。
注：由于 Exonuclease I 已经过充分失活处理，反应产物通常无需进行纯化处理，可直接用于后续相关实验；若后续实验对样品纯度要求较高，可以通过柱纯化、磁珠纯化，或者酚氯仿抽提、乙醇沉淀等方法纯化已降解单链 DNA 的反应产物。
6. 其它用途请自行根据实验目的，查阅相关文献资料进行。
常见问题：
1.PCR 反应产物通常会残留过量单链 DNA 引物，可以使用 Exonuclease I 进行单链引物清除吗？
可以的。Exonuclease I 最主要的应用就是消化 PCR 反应产物中或双链 DNA 样品中的多余单链 DNA。
2.Exonuclease I 可以催化双链 DNA 或 RNA 样品的酶切消除吗？
不可以。Exonuclease I 对作用底物具有高度选择性，只能催化 3'端为羟基的单链线性 DNA 的水解，对于其它类型的底物均没有催化酶解活性。
3.Exonuclease I 的最佳反应温度是多少？
Exonuclease I 在 37ºC 条件下具有最佳酶活和消化效果。
4.Exonuclease I 可以进行热失活吗？终止 Exonuclease I 酶活反应的条件是什么？
可以。反应完成后，将反应产物在 80ºC 条件下孵育 20 分钟即可使 Exonuclease I 充分失活，以终止反应。
5.Exonuclease I 在常规反应体系中是否都具有催化活性？
是的。Exonuclease I 的酶活在各种反应缓冲液中具有广谱的适应性，可以广泛适用于各种分子生物学实验体系中单链 DNA 的消化去除。
6.Exonuclease I 可用于将 3'端为突出粘性末端的双链 DNA 消化酶解生成平末端双链 DNA 吗？
不能。Exonuclease I 只能作用于单链 DNA，对于带有较短突出粘性末端的双链 DNA 不具备催化活性。
7.Exonuclease I 可以和碱性磷酸酶配合使用，用于在测序反应前或 PCR 反应后去除体系中多余的单链引物和 dNTP/NTP 吗？
可以。反应体系中同时加入 Exonuclease I 和碱性磷酸酶，可以在去除多余单链引物的同时，将 dNTP/NTP 进行去磷酸化失活处理。
8.Exonuclease I 是否可以和其它类型核酸外切酶配合使用，用于质粒样品的纯化？
Exonuclease I 可以和 Lambda Exonuclease 配合使用对质粒样品进行纯化制备。
9.Exonuclease I 与 RceJf Exonuclease 对之间有什么不同？
两者都是特异性作用于单链 DNA 分子的降解，但催化水解的方向相反，其中 Exonuclease I 是沿 3'→5'方向的消化单链 DNA 分子，而 RecJf Exonuclease 是沿 5'→3'方向酶解单链 DNA 分子。
10.Exonuclease I 能否沿 5'→3'方向降解单链 RNA 分子？
不能。Exonuclease I 是特异性作用于单链 DNA 的核酸外切酶，只能以单链 DNA 为底物，沿着 3'→5'方向催化单个脱氧核糖核苷酸的酶切和移除，而不能作用于 RNA 分子。
保存条件：
-20℃保存，两年有效，≤0℃运输。
注意事项：
（1）本产品提供的试剂均用不含 DNA 酶和 RNA 酶的水配制，可直接用于单链线性 DNA 从 3'到 5'方向的消化。
（2）Exonuclease I 对作用底物具有高度选择性，仅特异催化 3'末端为羟基的单链 DNA 从 3'到 5'方向的降解，不能催化 3'末端带有其它修饰基团的单链 DNA 的消化，也不能作用于双链 DNA 和 RNA。请务必选择特定类型的底物，使用 Exonuclease I 进行酶切反应。
（3）Exonuclease I 不适用于水解双链 DNA，对于含二级结构的单链 DNA 需变性才能被水解。
（4）Exonuclease I 的最佳反应温度是 37ºC，在 80ºC 条件下孵育 20 分钟可使其完全失活。
（5）Exonuclease I 在普通 PCR buffer 中也具有活性，适用于 PCR 产物中引物或其他单链的去除。
（6）酶使用时宜存放在冰盒内或冰浴上，使用完毕后宜立即放置于-20ºC 保存。
（7）本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
（8）为了您的安全和健康，请穿实验服并戴一次性手套操作。
Our Exonuclease I is recombinantly expressed from Escherichia coli. It is a 3'→5' exonuclease that specifically degrades single‑stranded linear DNA in the 3' to 5' direction. Exonuclease I exhibits high substrate specificity: it exclusively digests single‑stranded linear DNA with a free 3'-OH terminus in the 3'→5' direction, releasing individual dNMP residues progressively until only two deoxynucleotides remain at the 5' end. It cannot digest single‑stranded DNA with chemically blocked or modified 3' termini, nor does it hydrolyze double‑stranded DNA or RNA. Exonuclease I is commonly used to remove excess unincorporated single‑stranded DNA primers from PCR mixtures prior to downstream Sanger sequencing or SNP analysis. It is not suitable for blunting the 3' overhangs of double‑stranded DNA. Its primary function is the stepwise removal of individual nucleotides from single‑stranded DNA in the 3'→5' direction.
| Source | Recombinant expression in Escherichia coli
| | Appearance | Sterile liquid
| | Storage Buffer | 10mM Tris-HCl, 100mM NaCl, 5mM DTT, 0.5mM EDTA, 100µg/ml BSA, 50% Glycerol, pH7.5 @ 25ºC.
| | Enzyme Concentration | 20 U/μL | | Purity | Free of contaminating DNA/RNA endonucleases, other exonucleases, and phosphatases.
| | Activity Definition | One unit is defined as the amount of enzyme that will catalyze the release of 10nmol of acid-soluble nucleotide in a total reaction volume of 50μl in 30 minutes at 37 º C in 1X Exonuclease I Reaction Buffer with 0.17mg/ml single-stranded [3H]-DNA. |
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Components List
| E666128 | Components | Appearance | 2KU | 5×2KU | 25×2KU | Storage | | E666128A | Exonuclease I (20 U/µl) | Liquid | 100ul | 5×100µl | 25×100µl | -20℃. Avoid freeze/thaw cycle. | | E666128B | 10× Reaction Buffer | Liquid | 400µl | 5×400µl | 25×400µl | -20℃. Avoid freeze/thaw cycle. |
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Applications
Removal of single‑stranded primers from PCR products;
Elimination of residual single‑stranded DNA primers prior to Sanger sequencing and SNP genotyping;
Removal of linear single‑stranded DNA while preserving double‑stranded DNA;
Analysis of single‑stranded DNA with free 3'-hydroxyl termini.
Advantages
Exonuclease I efficiently removes excess single‑stranded DNA primers from PCR products before sequencing or further molecular analysis.
Protocol
1. Reaction system setup.
For the removal of single-stranded DNA from DNA samples, prepare the reaction system on ice according to the table below (take the 20 μL system as an example):
Reagent
| Volume
| Final Concentration
|
|---|
Ultrapure Water
| (17.5-X) µL | - | 10× Reaction Buffer
| 2 µL | 1X | | DNA Sample | X µL (100 nmol bases) | 5 nmol/µL | Exonuclease I (20 U/µl)
| 0.5 µL | 0.5 U/µL | Total Volume
| 20 µL | - |
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When removing single‑stranded DNA from PCR products, prepare the reaction system on ice referring to the table below (taking a 5 μL system as an example).
Reagent
| Volume
| Final Concentration
|
|---|
| PCR Product | 4.5 µL | - | Exonuclease I (20 U/µl)
| 0.5 µL | 2 U/µL | Total Volume
| 5 µL | - |
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Notes:
- The recommended DNA input in the table is relatively high; the sample volume can be significantly reduced when starting material is limited.
- Follow standard DNA handling protocols strictly. Use only DNase‑free reagents and consumables to avoid contamination.
- For optimal digestion efficiency, adjust substrate and enzyme dosage according to the unit definition to establish the optimal reaction condition.
- For multiple parallel reactions, premix all components except the DNA substrate, aliquot into tubes, then add substrate individually.
- Exonuclease I retains activity in most common PCR buffers and can be added directly to finished PCR products for primer removal.
- If digestion efficiency is poor in a custom buffer, use the supplied 10× Reaction Buffer for optimal performance.
2. Mixing and Centrifugation
Gently mix the assembled reaction system and centrifuge briefly to collect liquid at the bottom of the tube.
3. Incubation Condition
Incubate at 37 °C for 30 min; incubation time may be adjusted according to experimental requirements.
4. Reaction Termination
Heat inactivation at 80 °C for 20 min completely terminates Exonuclease I activity.
5. Downstream Processing
The treated product can be analyzed directly by polyacrylamide gel electrophoresis as required.Since the enzyme is fully heat‑inactivated, the reaction product is generally suitable for direct downstream experiments without further purification. For applications requiring high purity, purify by column purification, magnetic bead purification, phenol‑chloroform extraction, or ethanol precipitation.
6. Other Applications
Refer to relevant literature for other experimental purposes.
Frequently Asked Questions
- Can Exonuclease I remove residual single‑stranded primers from PCR products?Yes. Exonuclease I is widely used to digest excess single‑stranded primers in PCR products and double‑stranded DNA samples.
- Does Exonuclease I digest double‑stranded DNA or RNA?No. It is strictly specific for single‑stranded linear DNA with free 3'-OH termini; it has no activity toward other DNA forms or RNA.
- What is the optimal reaction temperature?The optimal reaction temperature of Exonuclease I is 37 °C.
- Can the enzyme be heat‑inactivated? What is the termination condition?Yes. Incubation at 80 °C for 20 min fully inactivates the enzyme.
- Is Exonuclease I active in common molecular biology buffers?Yes. It exhibits broad compatibility with various reaction buffers and can be used for single‑stranded DNA removal in most molecular biology workflows.
- Can it blunt 3' overhangs of double‑stranded DNA?No. Exonuclease I only acts on single‑stranded DNA and cannot trim short single‑stranded overhangs of duplex DNA.
- Can Exonuclease I be used with alkaline phosphatase to remove primers and degrade dNTPs/NTPs before sequencing?Yes. Combined use of Exonuclease I and alkaline phosphatase simultaneously removes single‑stranded primers and dephosphorylates residual dNTPs/NTPs.
- Can it be combined with other exonucleases for plasmid purification?Exonuclease I can be used together with Lambda Exonuclease for plasmid purification.
- What is the difference between Exonuclease I and RecJf Exonuclease?Both degrade single‑stranded DNA but in opposite directions: Exonuclease I digests in the 3'→5' direction, while RecJf Exonuclease acts in the 5'→3' direction.
- Can Exonuclease I degrade single‑stranded RNA in the 5'→3' direction?No. It is specific for single‑stranded DNA only and does not recognize or digest RNA.
Storage & Transportation
Store at −20 °C; valid for 2 years. Transport at ≤0 °C.
Precautions
- All reagents are prepared with DNase/RNase‑free water and can be directly used for 3'→5' digestion of single‑stranded linear DNA.
- Strict substrate specificity: only digests single‑stranded DNA with free 3'-OH ends; no activity toward modified 3' termini, double‑stranded DNA, or RNA. Select appropriate substrates carefully.
- Not applicable for double‑stranded DNA digestion; structured single‑stranded DNA requires denaturation prior to digestion.
- Optimal activity at 37 °C; complete inactivation by incubation at 80 °C for 20 min.
- Retains activity in standard PCR buffers, suitable for direct primer removal from PCR products.
- Keep the enzyme on ice during use and return promptly to −20 °C after use.
- For research use only. Not for clinical diagnosis, therapeutic use, food or pharmaceutical applications, or household storage.
- Wear laboratory coat and disposable gloves for personal safety during operation.
ZJ26F0737120
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