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基本描述
| 规格或纯度 | 医药级 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 英文名称 | T7 RNA Transcription Enzyme Mix | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 储存温度 | -20°C储存 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 超低温冰袋运输 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品描述 作为生物大分子，mRNA只能通过体外转录（IVT，in vitro transcription）的方法大规模合成，T7启动子是目前转录效率最高的启动子之一，因此采用T7 RNA聚合酶（T7 RNA Polymerase）进行体外转录可获得更多的合成产物。T7 RNA Transcription Enzyme Mix进行了转录反应体系的优化，从模板DNA T7启动子下游开始合成与DNA中一条链互补的RNA，简单快速获得大量的RNA分子。一个反应可以转录高达150-200μg的RNA，转录合成的RNA可用于诸如mRNA疫苗的制备、RNA结构与功能研究、RNA酶保护、探针杂交、RNAi、显微注射及体外翻译等多方面的下游应用。 T7 RNA Transcription Enzyme Mix组成原酶利用大肠杆菌大规模发酵表达，采用药用规格原辅料生产，并严格控制宿主蛋白质残留、核酸残留等，符合GMP规范的产品生产与质量管理规程保障生产过程及所有原辅料可追溯。 质量要求
遵循以下规范生产 1. ISO 9001:2015, certified facility。 2. 《GMP附录-细胞治疗产品》国家药品监督管理局。 3. 《人用基因治疗总论-中国药典2020》国家药典委。 4. USP Chapter <1043>, Ancillary Materials for Cell, Gene, and Tissue-Engineered Products用于细胞治疗，基因治疗和组织工程产品中的辅料。 5. USP Chapter <92>, Growth Factors and Cytokines Used in Cell Therapy Manufacturing 细胞治疗产品生产过程中细胞因子和生长因子。 6. Ph. Eur. General Chapter 5.2.12, Raw Materials of Biological Origin for the Production of Cell-based and Gene Therapy Medicinal Products用于生产细胞或基因治疗药物的生物来源原料。 ?产品用途 1) 合成单链RNA，用于mRNA疫苗制备；? 2) 合成高特异性RNA探针； 3) 合成siRNA前体；? 4) 制作RNA剪接反应（RNA splicing）的前体。 产品组成 T7RNA Transcription Enzyme mix, GMP Grade? 50μl 注意事项? 1. 模板效率和孵化时间 本试剂盒以1μg的模板投入量可以产生150-200μg的RNA，然而，不同模板的产量会因模板的序列、结构、长度、纯度以及特定RNA聚合酶启动子的序列和长度而有所不同。影响转录产量的污染物包括核糖核酸酶或污染物，如苯酚、微量金属和SDS。 2. 优化的反应: 推荐的反应条件可适用于大多数模板的体外转录，但是，对于某些模板，可以通过延长反应时间(4小时-过夜反应)，增加模板的用量来提高产率。 3. 模板含量： 下表总结了我们在调控模板数量方面的经验。结果可能因使用的模板而异，将反应时间延长至4-6小时可增加RNA的产量。
4. 保持RNase-free环境： 使用无RNase管和移液枪； 处理含有RNA的试剂盒组件或样品时应戴手套，并经常更换手套，特别是接触到RNase的潜在污染源，如门把手、钢笔、铅笔和人体皮肤后。 不使用时，应将所有试剂密封好。在孵育过程中，将所有含有RNA的试管密封。 5.? 由于10×Transcription Buffer浓度偏高，高盐环境会导致聚合酶失活，同时buffer中含有亚精氨成分，会与模板DNA形成沉淀，配制反应液时需调整组分加样顺序，计算好体系，先加水，然后加buffer，NTP，最后加模板和酶，以防止10×的高浓度盐离子对酶造成影响。 Product Description As a biological macromolecule, mRNA can only be synthesized on a large scale by in vitro transcription (IVT). T7 promoter is currently one of the most efficient transcriptional promoters, so T7 RNA Polymerase (T7 RNA Polymerase) is used. In vitro transcription can obtain more synthetic products. T7 RNA Transcription Enzyme Mix optimizes the transcription reaction system. It synthesizes RNA complementary to one strand of DNA from the downstream of the template DNA T7 promoter, and obtains a large number of RNA molecules simply and quickly. A reaction can transcribe up to 150-200μg of RNA. The synthesized RNA can be used for downstream in many aspects such as mRNA vaccine preparation, RNA structure and function research, RNase protection, probe hybridization, RNAi, microinjection, and in vitro translation. application. T7 RNA Transcription Enzyme Mix is ??composed of the original enzymes expressed by large-scale fermentation in Escherichia coli, produced with medicinal specifications raw materials, and strictly controlled host protein residues, nucleic acid residues, etc., in line with GMP standard product production and quality management procedures to ensure the production process and all The raw materials can be traced back. Quality requirements
Follow the following specifications for production 1. ISO 9001:2015, certified facility. 2. "GMP Appendix-Cell Therapy Products" State Drug Administration. 3. "General Introduction to Human Gene Therapy-Chinese Pharmacopoeia 2020" National Pharmacopoeia Commission. 4. USP Chapter <1043>, Ancillary Materials for Cell, Gene, and Tissue-Engineered Products are used as excipients in cell therapy, gene therapy and tissue engineering products. 5. USP Chapter <92>, Growth Factors and Cytokines Used in Cell Therapy Manufacturing Cytokines and growth factors used in the production of cell therapy products. 6. Ph. Eur. General Chapter 5.2.12, Raw Materials of Biological Origin for the Production of Cell-based and Gene Therapy Medicinal Products. ?Product Usage 1) Synthesize single-stranded RNA for mRNA vaccine preparation; 2) Synthesize highly specific RNA probes; 3) Synthesis of siRNA precursors; 4) Preparation of precursors for RNA splicing. Product composition T7RNA Transcription Enzyme mix, GMP Grade 50μl Precautions 1. Template efficiency and incubation time: This kit can produce 150-200μg RNA with 1μg template input. However, the yield of different templates will vary due to the sequence, structure, length, purity of the template, and the sequence and length of the specific RNA polymerase promoter. Contaminants that affect transcription yield include ribonuclease or contaminants such as phenol, trace metals, and SDS. 2. Optimized response: The recommended reaction conditions are suitable for in vitro transcription of most templates. However, for some templates, you can increase the yield by extending the reaction time (4 hours-overnight reaction) and increasing the amount of template. 3. Template content: The following table summarizes our experience in regulating the number of templates. The results may vary depending on the template used. Extending the reaction time to 4-6 hours can increase the RNA yield.
4. Maintain RNase-free environment: Use RNase-free tube and pipette; Wear gloves when handling kit components or samples containing RNA, and change gloves frequently, especially after contact with potential sources of RNase contamination, such as doorknobs, pens, pencils, and human skin. When not in use, all reagents should be sealed. During the incubation, all test tubes containing RNA are sealed. 5. Due to the high concentration of 10×Transcription Buffer, high-salt environment will cause polymerase inactivation. At the same time, the buffer contains spermidine, which will form a precipitate with the template DNA. When preparing the reaction solution, it is necessary to adjust the component addition order and calculate For a good system, add water first, then buffer, NTP, and finally template and enzyme to prevent the 10× high concentration of salt ions from affecting the enzyme. |
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