本公司生产的牛痘 DNA 拓扑异构酶 I，英文名为 Vaccinia DNA Topoisomerase I，也称牛痘病毒 DNA 拓扑异构酶 I (Vaccinia Virus DNA Topoisomerase I)，是一种来源于牛痘病毒(vaccinia virus)的 I 型真核拓扑异构酶，可以催化双链 DNA 分子中单链 DNA 特定序列位置的磷酸二酯 键的断开和连接。Vaccinia DNA Topoisomerase I 具有解超螺旋的酶(DNA Relaxing Enzyme)活性，可以通过快速的酶切和连接从而使双链共价 闭合环状的正或负超螺旋 DNA 发生解超螺旋，从而可以形成含有较少的正或负超螺旋的双链环状的 DNA 分子。Vaccinia DNA Topoisomerase I 也能使双链 DNA 形成绳结(knotting)或解开绳结(unknotting)，联结互补的单链环状 DNA 成为双链环状 DNA。Vaccinia DNA Topoisomerase I 目前常被作为一种 DNA 重组连接的新型工具酶，用于 DNA 载体和重组片段的连接、缺刻修复、接头连接等。Vaccinia DNA Topoisomerase I 特 异性识别的双链 DNA 中的 5'-… (C/T)CCTT…-3'序列如果位于距离 3'端几个碱基处，当 Vaccinia DNA Topoisomerase I 酶切 DNA 单链后，余下 的带有 5'羟基的单链 DNA 由于非常短而容易解离，此时 Vaccinia DNA Topoisomerase I 与 5'-… (C/T)CCTT…-3'序列中最后一个 T 的 3'磷酸基 团之间形成的共价复合物，也可以被其它的 5'端为羟基的 DNA 或 RNA 所攻击，从而形成重组的 DNA 分子或 DNA 与 RNA 的杂合分子。
纯度（Purity）：未检测到 Vaccinia DNA Topoisomerase I 活力之外的 DNA 外切酶、DNA 内切酶 和 RNase 活性。
组分和说明
| V752120 | Component | 200U | 5×200U
| 25×200U | Storage |
| V752120A | Vaccinia DNA Topoisomerase I (10U/µl) | 20µl | 5×20µl | 25×20µl | -20℃. 避免反复冻融。 |
| V752120B | 10× Reaction Buffer | 400μl | 5×400μl | 25×400μl | -20℃. 避免反复冻融。 |
| V752120C | Storage Buffer | 200µl | 5×200µl | 25×200μl
| -20℃. 避免反复冻融。 |
产品应用
Vaccinia DNA Topoisomerase I 可用于解开双链闭合环状 DNA 的超螺旋结构便于后续的酶切等分子生物学反应、DNA 载体和 PCR 扩增片段之间 的连接、缺刻修复和接头连接等。
产品优势
Vaccinia DNA Topoisomerase I 能够特异性识别双链 DNA 中 5'-… (C/T)CCTT…-3'序列，广泛的应用领域，可作为一种体外 DNA 连接的新型工 具，用于 DNA 载体和重组片段的连接、缺刻修复、接头连接等。
使用说明
1.溶解并混匀反应所需的各种溶液。将 Vaccinia DNA Topoisomerase I 置于冰浴上或冰盒内。
2.参考下表设置反应体系(以 20μl 体系为例)：
| Component | Volume | Final Concentration | | Nuclease-free Water | 15.9 μl | - | | 10X Reaction Buffer | 2 μl | 1X | | DNA (0.5μg/μl) | 2 μl | 50 ng/μl | | Vaccinia DNA Topoisomerase I (10U/µl) | 0.1 μl | 0.05 U/µl | | Total | 20 μl | - |
|
注：如果酶浓度过高，可适当使用本产品提供的 Storage Buffer 将酶适当稀释后再使用。
3.将上述配制好的反应体系置于 PCR 仪或水浴，37ºC 孵育 30min，然后放置冰上。
注：使用 PCR 仪孵育时，需要调整热盖温度为 40℃左右，避免在孵育过程中造成酶失活。
4.使用不含溴化乙锭(EtBr)的适当浓度的琼脂糖凝胶进行电泳，电泳结束后进行核酸染色和凝胶成像观察。
保存条件： -20℃保存，≤0℃运输
注意事项：
（1）当超螺旋质粒用 Vaccinia DNA Topoisomerase I 处理后，应使用不含溴化乙锭(EtBr，简称 EB)的琼脂糖凝胶进行电泳，必须在电泳完成后 再使用溴化乙锭或其它合适的核酸染料进行凝胶的核酸染色检测。因为当凝胶中含有溴化乙锭时，溴化乙锭会结合到 DNA 的碱基之间并催化形成 超螺旋。而在电泳结束后再进行染色，即使再次催化形成了超螺旋，也不会改变核酸片段的电泳位置了。
（2）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。
（3）为了您的安全和健康，请穿实验服并戴一次性手套操作。
Our Vaccinia DNA Topoisomerase I, also known as Vaccinia Virus DNA Topoisomerase I, is a type I eukaryotic topoisomerase derived from vaccinia virus. It catalyzes the cleavage and re-ligation of phosphodiester bonds at specific sequence positions on a single strand within double-stranded DNA molecules.
Vaccinia DNA Topoisomerase I exhibits DNA relaxing enzyme activity. Through rapid enzymatic cleavage and re-ligation, it relaxes positively or negatively supercoiled covalently closed circular double-stranded DNA, generating double-stranded circular DNA molecules with reduced positive or negative supercoiling. Vaccinia DNA Topoisomerase I can also knot or unknot double-stranded DNA, and join complementary single-stranded circular DNA to form double-stranded circular DNA. Currently, Vaccinia DNA Topoisomerase I is widely used as a novel tool enzyme for DNA recombination and ligation, for applications including ligation of DNA vectors and recombinant fragments, nick repair, and adaptor ligation.
If the 5'-…(C/T)CCTT…-3' double-stranded DNA sequence specifically recognized by Vaccinia DNA Topoisomerase I is located several bases away from the 3' end, after Vaccinia DNA Topoisomerase I cleaves the DNA single strand, the remaining short single-stranded DNA with a 5'-hydroxyl group readily dissociates. The covalent complex formed between Vaccinia DNA Topoisomerase I and the 3'-phosphate group of the final T in the 5'-…(C/T)CCTT…-3' sequence can be attacked by other 5'-hydroxyl-terminated DNA or RNA, yielding recombinant DNA molecules or DNA-RNA hybrid molecules.
Purity: No DNA exonuclease, DNA endonuclease or RNase activity other than the activity of Vaccinia DNA Topoisomerase I was detected.
Components & Description
V752120
| Component | 200U | 5×200U | 25×200U | Storage |
|---|
| V752120A | Vaccinia DNA Topoisomerase I (10U/µl) | 20µl | 5×20µl | 25×20µl | -20℃. Avoid freeze/thaw cycles. | | V752120B | 10× Reaction Buffer | 400μl | 5×400μl | 25×400μl | -20℃. Avoid freeze/thaw cycles. | | V752120C | Storage Buffer | 200µl | 5×200µl | 25×200µl | -20℃. Avoid freeze/thaw cycles. |
|
Product Applications
Vaccinia DNA Topoisomerase I can be used to relax the supercoiled structure of double-stranded closed circular DNA to facilitate subsequent molecular biological reactions such as restriction digestion, ligation between DNA vectors and PCR-amplified fragments, nick repair and adaptor ligation.
Product Advantages
Vaccinia DNA Topoisomerase I specifically recognizes the 5'-…(C/T)CCTT…-3' sequence in double-stranded DNA. With a broad range of applications, it serves as a novel tool for in vitro DNA ligation, for ligation of DNA vectors and recombinant fragments, nick repair, adaptor ligation and other related experiments.
Instructions for Use
1. Dissolve and mix all required solutions for the reaction. Keep Vaccinia DNA Topoisomerase I on ice or in an ice box.
2. Set up the reaction system referring to the table below (example for a 20 μl reaction):
| Component | Volume | Final Concentration |
|---|
| Nuclease-free Water | 15.9 μl | — |
| 10× Reaction Buffer | 2 μl | 1× |
| DNA (0.5 μg/μl) | 2 μl | 50 ng/μl |
| Vaccinia DNA Topoisomerase I (10U/µl) | 0.1 μl | 0.05 U/µl |
| Total | 20 μl | — |
Note: If the enzyme concentration is too high, dilute the enzyme appropriately with the provided Storage Buffer before use.
3. Incubate the prepared reaction mixture in a PCR instrument or water bath at 37°C for 30 min, then place on ice.
Note: When incubating with a PCR instrument, set the hot lid temperature to approximately 40°C to prevent enzyme inactivation during incubation.
4. Perform electrophoresis using agarose gel of appropriate concentration without ethidium bromide (EtBr). After electrophoresis, conduct nucleic acid staining and gel imaging observation.
Storage Conditions
Store at -20℃; transport at ≤0℃.
Precautions
(1) After supercoiled plasmid is treated with Vaccinia DNA Topoisomerase I, electrophoresis must be performed using agarose gel without ethidium bromide (EtBr). Nucleic acid staining with ethidium bromide or other suitable nucleic acid dyes must be carried out after electrophoresis. When ethidium bromide is present in the gel, EtBr intercalates between DNA bases and induces supercoiling. Staining after electrophoresis will not alter the electrophoretic migration position of nucleic acid fragments even if supercoiling is re-induced.
(2) This product is for scientific research use by professionals only. It shall not be used for clinical diagnosis or therapy, food or pharmaceutical applications, and shall not be stored in ordinary residences.
(3) Wear a lab coat and disposable gloves for your safety and health during operation.
ZJ25F0724897
危险品化学品经营许可证（带存储）