T7 RNA Transcription Enzyme Mix (C09-1073-855)

Product DescriptionAs 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 Project Standard Method Exterior Clear liquid Visual inspection Visible foreign body Compliance Chinese Pharmacopoeia 2020 Edition Fourth Part 1 Lamp Inspection Method (General Rule 0904) pH value 7.5-8.5 Chinese Pharmacopoeia 2020 Edition Part IV pH Determination Method (General Principle 0631) Performance 1μl Enzyme Mix product can transcribe no less than 100μg RNA RNA transcription yield method Endonuclease residue 004-DNA degradation does not exceed 10% Incubate 1μl enzyme with 004-DNA.37C for 3h Exonuclease residue 019 The degradation of HindⅢ DNA does not exceed 10%. 1μl enzyme and 019 HindⅢ DNA. Incubate at 37℃ for 3h RNase residue Degradation of 293-RNA does not exceed 10% 1μl Enzyme and 293-RNA. Incubate at 37°C for 1h Bacterial endotoxin content ≤10 EU/mg Chinese Pharmacopoeia 2020 Edition Fourth Gel Limit Test Method (General Rule 1143) Exogenous DNA residue ≤100 pg/mg Fluorescence quantitative PCR Host protein residue ≤50 ppm Chinese Pharmacopoeia 2020 Edition Part IV Method for the Determination of Bacterial Protein Residues (General Rule 3412) Mycoplasma detection Feminine Mycoplasma detection kit Heavy metal residue ≤10 ppm Chinese Pharmacopoeia 2020 Edition Fourth Heavy Metal Inspection Method (General Principle 0821) Follow the following specifications for production1. 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 Usage1) 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 compositionT7RNA Transcription Enzyme mix, GMP Grade 50μlPrecautions1. 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. Template amount RNA yield 1000ng(1μg) 130-160μg 500ng (0.5μg) 110-130μg 100ng (0.1μg) 30-50μg 50ng(0.05μg) 15-25μg 10ng(0.01μg) 10-20μg lng(0.001μg) 3-8μg 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. Specifications and Purity: Pharmaceutical grade.