本公司生产的E.coli Poly(A)加尾酶，即 E.coli Poly(A)聚合酶，也称 E.coli PAP、PAP 酶、Poly(A)加尾酶或 PolyA 加尾酶，能以不依赖于模板的方式催化由 ATP 转化成的 AMP 添加到单链 RNA 的 3’末端，以形成 Poly(A)尾。E.coli Poly(A)聚合酶能以各种单链 RNA 作为底物 RNA，但双链 RNA 及过短的寡核苷酸不推荐作为该反应的 RNA 底物，并且 DNA 不能作为该反应的底物。实测 15nt 合成的 RNA 完全可以作为 E.coli Poly(A) Polymerase 的底物，在 ATP 存在时催化形成 Poly(A)尾的。E.coli Poly(A) Polymerase 催化的 Poly(A)加尾反应只能使用 ATP，而不能使用 ADP 或 dATP。另外，使用 CTP 和 UTP 时，其掺入量不足使用 ATP 时的 5%；而使用 GTP 时，其完全不能添加到 RNA 的 3'末端。
| 来源（Source） | 大肠杆菌重组表达 | | 外观（Appearance） | 无菌液体 | | 保存液（Storage Buffer） | 20mM Tris-HCl (pH7.5, 25℃), 500mM NaCl, 1mM DTT, 1mM EDTA, 50% (v/v) Glycerol, 0.1% (w/v) Triton X-100 | | 酶浓度（Enzyme Concentration） | 5U/μL | | 纯度（Purity） | 不含 DNase、RNase 和磷酸酯酶 | | 活性定义（Activity Definition） | One unit is defined as the amount of enzyme that will incorporate 1nmol of AMP into RNA in a 20μl volume in 10 minutes at 37℃ |
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组分表
| P1522669 | 组件 | 物理外观 | 100U | 5×100U | 储存 | | P1522669A | E.coli Poly(A) 聚合酶 (5U/µl) | Liquid | 20µl | 5×20µl | -20℃. Avoid freeze/thaw cycle. | | P1522669B | 10× PAP 反应缓冲液 | Liquid | 50µl | 5×50µl | -20℃. Avoid freeze/thaw cycle. | | P1522669C | 10mM ATP | Liquid | 50µl | 5×50µl | -20℃. Avoid freeze/thaw cycle. |
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产品应用
用放射性同位素标记的 ATP 或虫草素(cordycepin)标记 RNA；为 RNA 添加 Poly(A)尾，用于反转录后克隆或亲和纯化；增加 mRNA 的稳定性，从而提高转染至真核细胞中的 RNA 的翻译效率。
产品优势
本产品能以不依赖于模板的方式催化由 ATP 转化成的 AMP 添加到单链 RNA 的 3’末端，以形成 Poly(A)尾。
使用说明
1.参考下表在冰浴中设置反应体系。 参考下表 20μl 反应体系中加 Poly(A)尾的 RNA 底物量可以高达 10μg，并且可根据实验需要按比例放大反应体系。在 Poly(A)加尾反应 中，加尾的长度取决于 RNA 3’-OH 末端的摩尔浓度、反应时间、酶量和 ATP 浓度。可以通过更改一个或多个因素来调整加尾的长度。按照如下的推荐反应体系，在 37℃孵育 30min，加 Poly(A)尾长度可以超过 100 个碱基。
| 试剂 | 体积 | 终浓度
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|---|
DEPC 处理水
| (14.5-x)µl | - | 10× PAP 反应缓冲液
| 2µl | 1X | | 10mM ATP | 2µl | 1mM | | RNase 抑制剂 (40U/µl) | 0.5µl | 1U/µl (可选) | | RNA | xµl | 0.05-0.5µg/µl | E.coli Poly(A) 聚合酶 (5U/µl)
| 1µl | 0.25U/µl | 总体积
| 20µl | - |
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注 1：由于涉及 RNA 操作，需要严格按照 RNA 操作的规范进行，避免 RNase 污染，相关试剂和耗材需要经过 DEPC 处理以去除 RNase 或者确保是 RNase free 的。
注 2：用于加尾反应的 RNA 在使用前应进行适当的纯化，并溶解于 nuclease free water 中，且溶液中应不含 EDTA 和盐分。
注 3：如果较难确保比较严格的 RNase-free，推荐在上述反应体系中添加 0.5µl RNase Inhibitor，以提高溶液中 RNA 的稳定性。
2.加尾反应：37℃孵育 30min。
3.反应终止：加入 EDTA 至终浓度为 10mM，或 65℃加热 20min 以终止反应。
保存条件：
-20℃保存，至少两年有效，≤0℃运输。
注意事项：
（1） 10× PAP 反应缓冲液中不包含 ATP。
（2） E.coli Poly(A) 聚合酶的用量主要影响目的产物的长度，可以根据所需目的产物的长度适当调整酶的用量，如果想得到长度较长的产物可以适当加大酶的用量或延长反应时间。
（3）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。
（4）为了您的安全和健康，请穿实验服并戴一次性手套操作。
The E. coli Poly(A) tailing enzyme produced by our company, also known as E. coli Poly(A) polymerase, E. coli PAP, PAP enzyme, Poly(A) tailing enzyme or PolyA tailing enzyme, catalyzes the template-independent addition of AMP derived from ATP to the 3'-end of single-stranded RNA to form a Poly(A) tail. E. coli Poly(A) polymerase can use various single-stranded RNAs as substrates. However, double-stranded RNA and excessively short oligonucleotides are not recommended as substrates for this reaction, and DNA cannot be used as a substrate. It has been experimentally verified that 15nt synthetic RNA can fully serve as a substrate for E. coli Poly(A) polymerase to catalyze the formation of a Poly(A) tail in the presence of ATP. The Poly(A) tailing reaction catalyzed by E. coli Poly(A) polymerase can only use ATP, not ADP or dATP. In addition, when using CTP or UTP, the incorporation efficiency is less than 5% of that with ATP; while GTP cannot be added to the 3'-end of RNA at all.
| Source | Recombinantly expressed in E. coli
| | Appearance | Sterile liquid
| | Storage Buffer | 20mM Tris-HCl (pH7.5, 25℃), 500mM NaCl, 1mM DTT, 1mM EDTA, 50% (v/v) Glycerol, 0.1% (w/v) Triton X-100 | | Enzyme Concentration | 5U/μL | | Purity | Free of DNase, RNase and phosphatase
| | Activity Definition | One unit is defined as the amount of enzyme that will incorporate 1nmol of AMP into RNA in a 20μl volume in 10 minutes at 37℃ |
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Components List
| P1522669 | Components | Appearance | 100U | 5×100U | Storage | | P1522669A | E.coli Poly(A) Polymerase (5U/µl) | Liquid | 20µl | 5×20µl | -20℃. Avoid freeze/thaw cycle. | | P1522669B | 10× PAP Reaction Buffer | Liquid | 50µl | 5×50µl | -20℃. Avoid freeze/thaw cycle. | | P1522669C | 10mM ATP | Liquid | 50µl | 5×50µl | -20℃. Avoid freeze/thaw cycle. |
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Product Applications
Labeling RNA with radioisotope-labeled ATP or cordycepin; Adding Poly(A) tails to RNA for post-reverse transcription cloning or affinity purification; Increasing mRNA stability to improve translation efficiency of RNA transfected into eukaryotic cells.
Product Advantages
This product catalyzes the template-independent addition of AMP derived from ATP to the 3'-end of single-stranded RNA to form a Poly(A) tail.
Instructions for Use
1. Set up the reaction system on ice according to the table below. The amount of RNA substrate for Poly(A) tailing in the 20μl reaction system can be up to 10μg, and the reaction system can be scaled up proportionally according to experimental needs. In the Poly(A) tailing reaction, the tail length depends on the molar concentration of the RNA 3'-OH end, reaction time, enzyme amount, and ATP concentration. The tail length can be adjusted by changing one or more of these factors. Using the recommended reaction system below and incubating at 37℃ for 30min, the Poly(A) tail length can exceed 100 bases.
| Reagent | Volume | Final Concentration |
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| DEPC-treated Water | (14.5-x)µl | - | | 10× PAP Reaction Buffer | 2µl | 1X | | 10mM ATP | 2µl | 1mM | | RNase Inhibitor (40U/µl) | 0.5µl | 1U/µl (Optional) | | RNA | xµl | 0.05-0.5µg/µl | | E.coli Poly(A) Polymerase (5U/µl) | 1µl | 0.25U/µl | | Total Volume | 20µl | - |
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Note 1: Since RNA handling is involved, strictly follow RNA operation specifications to avoid RNase contamination. Relevant reagents and consumables should be treated with DEPC to remove RNase or be confirmed as RNase-free.
Note 2: RNA used for the tailing reaction should be properly purified before use, dissolved in nuclease-free water, and the solution should contain no EDTA or salts.
Note 3: If strict RNase-free conditions are difficult to ensure, it is recommended to add 0.5µl RNase Inhibitor to the above reaction system to improve RNA stability in the solution.
2. Tailing reaction: Incubate at 37℃ for 30min.
3. Reaction termination: Stop the reaction by adding EDTA to a final concentration of 10mM, or heating at 65℃ for 20min.
Storage Conditions
Store at -20℃, valid for at least 2 years. Transport at ≤0℃.
Precautions
(1) The 10× PAP Reaction Buffer does not contain ATP.
(2) The amount of E. coli Poly(A) Polymerase mainly affects the length of the target product. The enzyme amount can be adjusted according to the desired product length. To obtain longer products, appropriately increase the enzyme amount or extend the reaction time.
(3) This product is for scientific research use only by professionals. It shall not be used for clinical diagnosis or treatment, food or pharmaceutical applications, nor stored in ordinary residential premises.
(4) For your safety and health, wear a lab coat and disposable gloves when handling.
危险品化学品经营许可证（带存储）