本公司生产的T4 RNA 连接酶 2 (T4 RNA Ligase 2)，是一种 ATP 依赖的双链 RNA 连接酶(double-strand RNA ligase, dsRNA Ligase)，也被称 为 T4 Rnl2 (gp24.1)，可以用于双链 RNA 进行分子内的环化连接和分子间的线性连接。T4 RNA Ligase 2 与 T4 RNA Ligase 1 不同的是，对双链 RNA 中的缺刻(nick)的连接活性要大大高于对单链 RNA 末端的连接活性。T4 RNA Ligase 2 也可用于双链核酸(RNA 双链、RNA/DNA 杂合链和/ 或 DNA 双链)分子内或分子间 RNA 链的 3'羟基和 DNA 链的 5'磷酸基的连接。T4 RNA Ligase 2 连接时需要 5'磷酸和 3'羟基的存在，可以是 RNA 链或 DNA 链的 5'磷酸基和 RNA 链的 3'羟基之间发生连接反应。T4 RNA Ligase 2 催化 dsRNA 粘端连接的反应过程如下。首先 T4 RNA Ligase 2 直接消耗 ATP，形成中间产物 T4 RNA Ligase 2-AMP，并释放焦磷酸；接着，T4 RNA Ligase 2-AMP 与 dsRNA 的缺刻处相结合，并将 AMP 从中间产物 T4 RNA Ligase 2-AMP 中转移至 dsRNA 缺刻处的 5'磷酸末端，形成腺苷酰化缺刻 dsRNA 中间产物；最后，在 T4 RNA Ligase 2 的催化下， dsRNA 缺刻处的 3'羟基进攻该缺刻处的 5'磷酸基团，形成 3'-5'磷酸二酯键，并释放出 AMP。
| 项目 | 参数 |
|---|
| 来源（Source） | 大肠杆菌重组表达 | | 外观（Appearance） | 无菌液体 | | 保存液（Storage Buffer） | 10 mM Tris-HCl, 50 mM KCl, 35 mM (NH₄)₂SO₄, 0.1 mM EDTA, 0.1 mM DTT, 50% (v/v) Glycerol, pH7.5.
| | 酶浓度（Enzyme Concentration） | 10U/μL | | 纯度（Purity） | 不含 DNA 内切酶和外切酶，不含 RNA 酶，不含磷酸酯酶。 | | 活性定义（Activity Definition） | One unit is defined as the amount of enzyme required to ligate 0.4 μg of an equimolar mix of a 23-mer and 17-mer RNAs in a total reaction volume of 20 μl in 30 minutes at 37℃. |
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组分表
T750873
| 组件 | 物理外观 | 1000U | 5×1000U
| 储存 | | T750873A | T4 RNA 连接酶 2 (10U/µl) | 液体 | 100ul | 5×100ul
| -20℃. 避免反复冻融。 | | T750873B | 10×T4 RNA 连接酶 2 反应缓冲液
| 液体 | 250ul | 5×250ul | -20℃. 避免反复冻融。 |
|
产品应用
主要用于连接双链 RNA 中缺刻的连接(即双链 RNA 的粘端连接)，也可用于双链结构中，RNA 3'羟基与 DNA 5'磷酸基的缺刻连接。
产品优势
本产品酶活性高，连接效率高。
使用说明
1. 双链 RNA 或 DNA/RNA 杂合双链的退火。 将两条单链 RNA 等摩尔数混合，推荐的终浓度为 20µM (10-50µM 均可)，90℃孵育 1min，然后通过梯度降温至 25℃退火形成 dsRNA。推荐使用 Annealing Buffer for RNA oligos (5×)，并按照该产品使用说明进行退火反应。DNA 和 RNA 杂合链的退火也可以参考双链 RNA 的退火条件进行。退火后的双链推荐在-80℃保存。
2. 对于双链 RNA 缺刻的连接，参考下表在冰浴中配制如下反应体系：
| 试剂名称 | 加入体积 | 反应体系终浓度 |
|---|
| DEPC 处理水 | 16μl | - | | Nicked dsRNA 底物（20μM） | 1μl | 1μM | | 10×T4 RNA 连接酶 2 反应缓冲液 | 2μl | 1× | | T4 RNA 连接酶 2（10U/μl） | 1μl | 0.5U/μl | | 反应体系总体积 | 20μl | - |
|
注意：
a.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 处理去除 RNase 或者确保是 RNase free 的。本反应体系中涉及双链 RNA，双链 RNA 可以耐受 RNase A 和 T1 等。如果涉及单链 RNA，包括双链退火后存在部分 RNA 序列是单链的情况，推荐适量添加 RNase Inhibitor。
b.如果同时进行多个连接反应，可以把上表中除 Nicked dsRNA 底物之外的所有溶液和酶提前预混合，然后再分装到各反应管内。
c.反应体系中的 Nicked dsRNA 底物的最终浓度可以达到 1µM，在该反应体系中可以确保充分连接。实际使用过程中，例如由于底物量有限等原因，可以适当减少 Nicked dsRNA 底物的用量，例如使最终浓度为 0.5µM 或 0.2µM 等。
3. 对于 RNA/DNA 杂合链中 RNA 3'羟基与 DNA 5'磷酸基的缺刻连接，参考下表在冰浴中配制如下反应体系：
| 试剂名称 | 加入体积 | 反应体系终浓度 |
|---|
| DEPC 处理水 | 10.4μl | - | | Nicked DNA/RNA 底物（20μM） | 1μl | 1μM | | 10×T4 RNA 连接酶 2 反应缓冲液 | 2μl | 1× | | PEG8000（50%，无 RNase） | 4μl | 0.1 | MgCl₂ (100mM, DEPC 处理)
| 1.6μl | 8mM | | T4 RNA 连接酶 2（10U/μl） | 1μl | 0.5U/μl | | 反应体系总体积 | 20μl | - |
|
注意：
a.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 处理去除 RNase 或者确保是 RNase free 的。本反应体系中涉及双链 RNA，双链 RNA 可以耐受 RNase A 和 T1 等。如果涉及单链 RNA，包括双链退火后存在部分 RNA 序列是单链的情况，推荐适量添加 RNase Inhibitor。
b.如果同时进行多个连接反应，可以把上表中除 Nicked DNA/RNA 底物之外的所有溶液和酶提前预混合，然后再分装到各反应管内。
c.反应体系中的 Nicked dsRNA 底物的最终浓度可以达到 1µM，在该反应体系中可以确保充分连接。实际使用过程中，例如由于底物量有限等原因，可以适当减少 Nicked dsRNA 底物的用量，例如使最终浓度为 0.5µM 或 0.2µM 等。
4. 连接反应：37℃孵育 30min。如果发现效果欠佳可以尝试 25℃孵育 2h。为了使连接反应更加充分，可以适当延长连接反应时间。
5. 终止反应：反应结束后加入蛋白酶 K 或 EDTA，即可终止反应。 由于 T4 RNA Ligase 2 热失活需要 85℃加热 5min，这样可能导致 dsRNA 或 DNA/RNA 杂合双链变性，因此通常不建议通过加热方式失活 T4 RNA Ligase 2。如果后续无需保持双链状态，则推荐 85℃加热 5min 以失活 T4 RNA Ligase 2。
注意事项：
（1）如果连接底物为 ssRNA，推荐使用 T4 RNA Ligase 1 等系列产品。
（2）可根据具体应用选择合适的操作方法，可能需准备额外的试剂，如 RNase Inhibitor、DEPC 水等。
（3）本产品提供的T4 RNA 连接酶 2 反应缓冲 (10×)适用于 RNA 双链中缺刻的连接反应。如果用于 RNA/DNA 杂合链中 RNA 3'羟基与 DNA 5'磷酸 基的缺刻连接时，需要将反应体系中的 MgCl₂ 的终浓度提高至 10mM，并在反应体系中加入终浓度为 10-15%的 PEG8000，这样可以显著提高酶 活性，同时不影响反应特性。
（4）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。
（5）为了您的安全和健康，请穿实验服并戴一次性手套操作。
T4 RNA Ligase 2 is an ATP-dependent double-strand RNA ligase (dsRNA Ligase), also called T4 Rnl2 (gp24.1). It can be used for intramolecular circular ligation and intermolecular linear ligation of double-stranded RNA.Different from T4 RNA Ligase 1, T4 RNA Ligase 2 has much higher ligation activity for nicks in double-stranded RNA than for the termini of single-stranded RNA. T4 RNA Ligase 2 can also be used for intramolecular or intermolecular ligation between the 3'-hydroxyl group of RNA strands and the 5'-phosphate group of DNA strands in duplex nucleic acids (double-stranded RNA, RNA/DNA heteroduplexes and/or double-stranded DNA).The ligation reaction of T4 RNA Ligase 2 requires the presence of 5'-phosphate and 3'-hydroxyl groups; the ligation reaction can occur between the 5'-phosphate group of an RNA or DNA strand and the 3'-hydroxyl group of an RNA strand.The reaction process of dsRNA cohesive-end ligation catalyzed by T4 RNA Ligase 2 is as follows. Firstly, T4 RNA Ligase 2 directly consumes ATP to form the intermediate T4 RNA Ligase 2-AMP and release pyrophosphate; secondly, T4 RNA Ligase 2-AMP binds to the nick site of dsRNA, and transfers AMP from the intermediate T4 RNA Ligase 2-AMP to the 5'-phosphate terminus at the dsRNA nick to form an adenylated nicked dsRNA intermediate; finally, under the catalysis of T4 RNA Ligase 2, the 3'-hydroxyl group at the dsRNA nick attacks the 5'-phosphate group at the same nick, forming a 3'-5' phosphodiester bond and releasing AMP.
| Item | Content |
|---|
| Source | Recombinantly expressed in Escherichia coli | | Appearance | Sterile liquid | | Storage Buffer | 10 mM Tris-HCl, 50 mM KCl, 35 mM (NH₄)₂SO₄, 0.1 mM EDTA, 0.1 mM DTT, 50% (v/v) Glycerol, pH7.5.
| | Enzyme Concentration | 10 U/μL | | Purity | Free of DNA endonuclease, DNA exonuclease, RNase and phosphatase | | Activity Definition | One unit is defined as the amount of enzyme required to ligate 0.4 μg of an equimolar mix of a 23-mer and 17-mer RNAs in a total reaction volume of 20 μL within 30 minutes at 37 °C. |
|
Components List
T750873
| Components
| Appearance
| 1000U | 5×1000U
| Storage
| | T750873A | T4 RNA Ligase 2 (10U/µl) | Liquid | 100ul | 5×100ul
| -20℃. Avoid freeze/thaw cycle. | | T750873B | 10× T4 Rnl2 Reaction Buffer
| Liquid
| 250ul | 5×250ul | -20℃. Avoid freeze/thaw cycle. |
|
Applications
It is mainly used for ligation of nicks in double-stranded RNA (i.e., cohesive-end ligation of dsRNA). It can also catalyze nick ligation between the 3'-hydroxyl of RNA and the 5'-phosphate of DNA within duplex structures.
Product Advantages
This product features high enzymatic activity and superior ligation efficiency.
Instructions for Use
1. Annealing of double-stranded RNA or DNA/RNA heteroduplexes. Mix two single-stranded RNAs at equimolar ratios; the recommended stock concentration is 20 μM (10–50 μM is acceptable). Incubate at 90 °C for 1 min, then perform gradient cooling down to 25 °C to form dsRNA. It is recommended to use 5× Annealing Buffer for RNA oligos and carry out annealing reactions following the manufacturer’s instructions of this buffer. Annealing conditions for DNA/RNA heteroduplexes can refer to those for double-stranded RNA. The annealed duplexes are recommended to be stored at -80 °C.
2. For ligation of nicks in double-stranded RNA, prepare the following reaction system on ice according to the table below:
| Reagent | Volume | Final Concentration |
|---|
| DEPC-treated Water | 16 μL | - | | Nicked dsRNA Substrate (20 μM) | 1 μL | 1 μM | | 10× T4 Rnl2 Reaction Buffer | 2 μL | 1× | | T4 RNA Ligase 2 (10 U/μL) | 1 μL | 0.5 U/μL | | Total Volume | 20 μL | - |
|
Notes:a. Since RNA manipulation is involved, standard RNA handling protocols must be strictly followed to prevent RNase contamination. All relevant reagents and consumables shall be treated with DEPC to remove RNase or certified RNase-free. This reaction system contains double-stranded RNA, which is resistant to RNase A, RNase T1 and other ribonucleases. If single-stranded RNA is present, including partially single-stranded RNA regions formed after duplex annealing, an appropriate amount of RNase Inhibitor is recommended to be supplemented.
b. When setting up multiple ligation reactions at the same time, pre-mix all solutions and enzymes listed in the above table except the Nicked dsRNA Substrate in advance, then aliquot the mixture into separate reaction tubes.
c. The final concentration of Nicked dsRNA Substrate in the reaction system can reach 1 µM, a concentration sufficient to guarantee complete ligation. In practical applications, the dosage of Nicked dsRNA Substrate can be appropriately reduced if substrate is limited; for instance, the final concentration can be adjusted to 0.5 µM or 0.2 µM.
3. For nick ligation between the 3'-hydroxyl group of RNA and the 5'-phosphate group of DNA within RNA/DNA heteroduplexes, prepare the reaction system on ice as specified in the table below:
| Reagent | Volume | Final Concentration |
|---|
| DEPC-treated Water | 10.4μl | - | | Nicked DNA/RNA Substrate (20μM) | 1μl | 1μM | | 10X T4 Rnl2 Reaction Buffer | 2μl | 1X | | PEG8000 (50%, RNase Free) | 4μl | 0.1 | | MgCl₂ (100mM, DEPC-treated) | 1.6μl | 8mM | | T4 RNA Ligase 2 (10U/μl) | 1μl | 0.5U/μl | | Total Volume | 20μl | - |
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Notes:
a. Since RNA handling is involved, standard RNA operation specifications must be strictly followed to avoid RNase contamination. All relevant reagents and consumables shall be treated with DEPC to eliminate RNase or be verified RNase-free. This reaction system contains double-stranded RNA, which can resist RNase A, RNase T1 and other ribonucleases. If single-stranded RNA exists, including partially single-stranded RNA regions formed after duplex annealing, appropriate amount of RNase Inhibitor is recommended to be added.b. When setting up multiple ligation reactions simultaneously, pre-mix all solutions and enzymes listed in the above table except the Nicked DNA/RNA Substrate in advance, then aliquot the mixture into individual reaction tubes.c. The final concentration of Nicked dsRNA Substrate in the reaction system can reach 1 µM, which can ensure complete ligation. In practical use, if the substrate is insufficient, the dosage of Nicked dsRNA Substrate can be reduced appropriately, for example, adjusting the final concentration to 0.5 µM or 0.2 µM.
4. Ligation reaction: Incubate at 37 °C for 30 min. If unsatisfactory ligation efficiency is observed, incubation at 25 °C for 2 h can be attempted. The incubation time can be extended appropriately to achieve more sufficient ligation.
5. Termination of reaction: The reaction can be terminated by adding Proteinase K or EDTA after incubation. T4 RNA Ligase 2 requires heating at 85 °C for 5 min for thermal inactivation, which may denature dsRNA or DNA/RNA heteroduplexes. Therefore, thermal inactivation of T4 RNA Ligase 2 is generally not recommended. If maintenance of duplex structure is not required for subsequent experiments, heating at 85 °C for 5 min is suggested to inactivate T4 RNA Ligase 2.
Precautions:
(1) If the ligation substrate is ssRNA, products such as T4 RNA Ligase 1 are recommended.
(2) Appropriate operating procedures can be selected according to specific applications, and extra reagents such as RNase Inhibitor and DEPC-treated Water may be required.
(3) The supplied 10× T4 Rnl2 Reaction Buffer is suitable for nick ligation within double-stranded RNA. For nick ligation between the 3'-hydroxyl of RNA and the 5'-phosphate of DNA in RNA/DNA heteroduplexes, the final concentration of MgCl₂ in the reaction system shall be increased to 10 mM, and PEG8000 shall be supplemented to a final concentration of 10–15%. This modification can markedly enhance enzymatic activity without altering reaction characteristics.
(4) This product is only for scientific research use by professional personnel. It shall not be used for clinical diagnosis or treatment, food or pharmaceutical manufacturing, nor stored in residential premises.
(5) Please wear lab coat and disposable gloves during operation for your safety and health.
ZJ26F0938953
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