{"product_id":"t7-high-yield-rna-synthesis-kit-bht20800006","title":"T7 High Yield RNA Synthesis Kit","description":"\u003cdiv class=\"product-block-list__item product-block-list__item--description\"\u003e\n                    \u003cdiv class=\"card\"\u003e\n\u003cdiv class=\"card__header\"\u003e\n                          \u003ch2 class=\"card__title heading h3\"\u003eDescription\u003c\/h2\u003e\n                        \u003c\/div\u003e\n\n                        \u003cdiv class=\"card__section \"\u003e\n                          \u003cdiv class=\"rte text--pull\"\u003e\n                            \u003cdiv class=\"top_tabc proDetail\" data-v-444b0c7e=\"\"\u003e\n\u003cdiv data-v-444b0c7e=\"\"\u003e\n\u003cdiv data-v-444b0c7e=\"\"\u003e\n\u003cp\u003eT7 High Yield RNA Synthesis Kit optimizes the transcription reaction system. The kit can synthesize the single-stranded RNA efficiently by using T7 RNA polymerase, the linear double-stranded DNA with the T7 promoter sequence as the template, NTPs as the substrate to control the DNA sequence downstream of the promoter. During transcription, modified nucleotides can be added to the substrate to prepare biotin or dye-labeled RNA.\u003c\/p\u003e\n\u003cp\u003eThis kit can synthesize long transcripts and short transcripts, RNA can be produced 100-200 μg with 1 μg of DNA template input. The RNA synthesized by transcription can be used for various downstream applications, such as RNA structure and function research, RNase protection, probe hybridization, RNAi, microinjection, and in vitro translation.\u003c\/p\u003e\n\u003cp\u003e\u003cimg src=\"https:\/\/seas.ysbuy.com\/file\/6794\/8909-167029822101711082.png\"\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan\u003eFigure 1: In vitro RNA transcription process\u003c\/span\u003e\u003c\/strong\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeature\u003c\/strong\u003e\u003c\/p\u003e\n\u003cul\u003e\n\u003cli\u003eUp to 180 μg of RNA per reaction from 1 μg of the control template\u003c\/li\u003e\n\u003cli\u003eOptimized reaction system for the IVT process\u003c\/li\u003e\n\u003cli\u003eDecrease the dsRNA production\u003c\/li\u003e\n\u003cli\u003eHigher RNA integrity and purity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003cp\u003e\u003cstrong\u003eApplication\u003c\/strong\u003e\u003c\/p\u003e\n\u003cul\u003e\n\u003cli\u003eIn vitro RNA synthesis\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003ch3 class=\"H3_backgro\"\u003e\u003cspan\u003e\u003cstrong\u003eComponents\u003c\/strong\u003e\u003c\/span\u003e\u003c\/h3\u003e\n\u003cdiv class=\"tableResponsive\"\u003e\n\u003ctable cellspacing=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd\u003eComponents No.\u003c\/td\u003e\n\u003ctd\u003eName\u003c\/td\u003e\n\u003ctd\u003e10623ES50 (50 T)\u003c\/td\u003e\n\u003ctd\u003e10623ES60 (100 T)\u003c\/td\u003e\n\u003ctd\u003e10623ES70 (500 T)\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e10623-A\u003c\/td\u003e\n\u003ctd\u003eT7 RNA Polymerase Mix\u003c\/td\u003e\n\u003ctd\u003e100 μL\u003c\/td\u003e\n\u003ctd\u003e200 μL\u003c\/td\u003e\n\u003ctd\u003e1 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e10623-B\u003c\/td\u003e\n\u003ctd\u003e10×Transcription Buffer\u003c\/td\u003e\n\u003ctd\u003e100 μL\u003c\/td\u003e\n\u003ctd\u003e200 μL\u003c\/td\u003e\n\u003ctd\u003e1 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e10623-C\u003c\/td\u003e\n\u003ctd\u003eATP (100mM)\u003c\/td\u003e\n\u003ctd\u003e100 μL\u003c\/td\u003e\n\u003ctd\u003e200 μL\u003c\/td\u003e\n\u003ctd\u003e1 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e10623-D\u003c\/td\u003e\n\u003ctd\u003eCTP (100mM)\u003c\/td\u003e\n\u003ctd\u003e100 μL\u003c\/td\u003e\n\u003ctd\u003e200 μL\u003c\/td\u003e\n\u003ctd\u003e1 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e10623-E\u003c\/td\u003e\n\u003ctd\u003eGTP (100mM)\u003c\/td\u003e\n\u003ctd\u003e100 μL\u003c\/td\u003e\n\u003ctd\u003e200 μL\u003c\/td\u003e\n\u003ctd\u003e1 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e10623-F\u003c\/td\u003e\n\u003ctd\u003eUTP (100mM)\u003c\/td\u003e\n\u003ctd\u003e100 μL\u003c\/td\u003e\n\u003ctd\u003e200 μL\u003c\/td\u003e\n\u003ctd\u003e1 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e10623-G\u003c\/td\u003e\n\u003ctd\u003eControl DNA Template (500ng\/μL)\u003c\/td\u003e\n\u003ctd\u003e10 μL\u003c\/td\u003e\n\u003ctd\u003e20 μL\u003c\/td\u003e\n\u003ctd\u003e100 μL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003ch3 class=\"H3_backgro\"\u003e\u003cspan\u003e\u003cstrong\u003eShipping and Storage\u003c\/strong\u003e\u003c\/span\u003e\u003c\/h3\u003e\n\u003cp\u003eDry ice transportation. Store at -15℃ ~ -25℃, valid for two years.\u003c\/p\u003e\n\u003ch3 class=\"H3_backgro\"\u003e\u003cspan\u003e\u003cstrong\u003eFigures\u003c\/strong\u003e\u003c\/span\u003e\u003c\/h3\u003e\n\u003cp\u003e\u003cimg src=\"https:\/\/seas.ysbuy.com\/file\/442040\/54b7-167029833660496583.png\"\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003eFigure 1. Standard RNA was synthesized in vitro using T7 RNA synthesis kit.\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003eThe reaction was incubated in a PCR instrument at 37℃ for 2h and then purified by magnetic beads (Cat#12602). The yield result was analyzed by NanoDrop spectrophotometer as shown in Figure 1.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cimg src=\"https:\/\/seas.ysbuy.com\/file\/98179\/18d6-167029881244617985.png\" alt=\"\"\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003eFigures 2. The transcription demonstration of different lengths of RNA by T7 kit respectively in the electrophoretogram (Figures 2A), the capillary electrophoresis diagram (Figures 2B), and the chromatogram (Figures 2C)\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cimg alt=\"\" src=\"https:\/\/cdn.shopifycdn.net\/s\/files\/1\/0803\/9419\/1166\/files\/T7_480x480.png?v=1700873194\"\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003eFigure 3. Synthesis of capped RNA in vitro.\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003eThe reaction was incubated in PCR instrument at 37℃ for 2h, and then purified by magnetic beads (Cat#12602). The yield result was assayed by NanoDrop spectrophotometer as shown in Figure 3A. The integrity result was analyzed by capillary electrophoresis as shown in Figure 3B.\u003c\/span\u003e\u003c\/p\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"top_tabc\" data-v-444b0c7e=\"\"\u003e\n\u003cspan class=\"boxTap\" id=\"tabCBox1\" data-v-444b0c7e=\"\"\u003eDocuments:\u003c\/span\u003e\n\u003cdiv data-v-444b0c7e=\"\"\u003e\n\u003cdiv class=\"cwrap\" data-v-444b0c7e=\"\" data-v-98c835fc=\"\"\u003e\n\u003cdiv data-v-98c835fc=\"\"\u003e\n\u003cdiv class=\"list\" data-v-98c835fc=\"\"\u003e\u003cbr\u003e\u003c\/div\u003e\n\u003cdiv class=\"list\" data-v-98c835fc=\"\"\u003e\u003cbr\u003e\u003c\/div\u003e\n\u003cdiv class=\"list\" data-v-98c835fc=\"\"\u003e\n\u003cdiv data-v-98c835fc=\"\"\u003e\n\u003cdiv class=\"title\" data-v-98c835fc=\"\"\u003eManuals\u003c\/div\u003e\n\u003cdiv data-v-98c835fc=\"\"\u003e\n\u003cp data-v-98c835fc=\"\"\u003e\u003ca href=\"https:\/\/cdn.shopify.com\/s\/files\/1\/0803\/9419\/1166\/files\/10623_Manual_HB230115_EN.pdf?v=1740621738\"\u003e10623_Manual_HB20230115\u003c\/a\u003e\u003c\/p\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"list\" data-v-98c835fc=\"\"\u003e\u003cbr\u003e\u003c\/div\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"top_tabc\" data-v-444b0c7e=\"\"\u003e\n\u003cspan class=\"boxTap\" id=\"tabCBox2\" data-v-444b0c7e=\"\"\u003eFAQs:\u003c\/span\u003e\n\u003cdiv data-v-444b0c7e=\"\"\u003e\n\u003cdiv data-v-444b0c7e=\"\"\u003e\n\u003ch3 class=\"H3_backgro\"\u003e\u003cstrong\u003e1. Low transcript yield\u003c\/strong\u003e\u003c\/h3\u003e\n\u003cp\u003eThe quality of the template is closely related to the yield. If the yield of the experimental group is significantly lower than the control group, the possible reasons are: \u003c\/p\u003e\n\u003cp\u003e① the experimental template contains inhibitory components; \u003c\/p\u003e\n\u003cp\u003e② The template has something wrong.\u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuggestions: \u003c\/strong\u003e\u003c\/p\u003e\n\u003cp\u003e① Re-purify the template; \u003c\/p\u003e\n\u003cp\u003e② Determine the template quantification and its integrity; \u003c\/p\u003e\n\u003cp\u003e③ Extend the reaction time; \u003c\/p\u003e\n\u003cp\u003e④ Increase the amount of template input; \u003c\/p\u003e\n\u003cp\u003e⑤ Try other promoters and RNA polymerases.\u003c\/p\u003e\n\u003ch3 class=\"H3_backgro\"\u003e\u003cstrong\u003e2. Low yield of short transcripts\u003c\/strong\u003e\u003c\/h3\u003e\n\u003cp\u003eA short transcription initiation fragment will inhibit the reaction. When the transcription product is less than 100 nt, extending the reaction time to 4-8 hs or increasing the amount of template to 2 μg will increase RNA yield.\u003c\/p\u003e\n\u003ch3 class=\"H3_backgro\"\u003e\u003cstrong\u003e3. RNA transcription length is greater than expected\u003c\/strong\u003e\u003c\/h3\u003e\n\u003cp\u003eIf the electrophoresis shows that the product band is larger than the expected size, the possible reasons: \u003c\/p\u003e\n\u003cp\u003e①The plasmid template may not be completely linearized; \u003c\/p\u003e\n\u003cp\u003e②The 3' end of the sense strand has a prominent structure; \u003c\/p\u003e\n\u003cp\u003e③The RNA has a secondary structure that is not completely denatured.\u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuggestions: \u003c\/strong\u003e\u003c\/p\u003e\n\u003cp\u003e①Check whether the template is completely linearized, and if necessary, perform additional linearization; \u003c\/p\u003e\n\u003cp\u003e②Select a suitable restriction enzyme to avoid 3' overhangs, or use Klenow Fragment \/T4 DNA polymerase to complete the transcription before proceeding; \u003c\/p\u003e\n\u003cp\u003e③Use denatured gel to detect RNA products.\u003c\/p\u003e\n\u003ch3 class=\"H3_backgro\"\u003e\u003cstrong\u003e4. RNA transcription length is less than expected\u003c\/strong\u003e\u003c\/h3\u003e\n\u003cp\u003eIf the electrophoresis shows that the product band is smaller than the expected size, the possible reasons: \u003c\/p\u003e\n\u003cp\u003e①The template contains a termination sequence similar to T7 RNA polymerase;\u003c\/p\u003e\n\u003cp\u003e②The GC content in the template is high.\u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuggestions: \u003c\/strong\u003e\u003c\/p\u003e\n\u003cp\u003e①Lower the reaction temperature (for example, 30°C). Sometimes lowering the temperature can increase the transcription length, but it will reduce the yield. Or try different RNA polymerases for transcription; \u003c\/p\u003e\n\u003cp\u003e②If the template GC content is high, use 42℃ to transcript, or add SSB to increase the yield and transcription length.\u003c\/p\u003e\n\u003ch3 class=\"H3_backgro\"\u003e\u003cstrong\u003e5. Electrophoresis tailing of transcription products\u003c\/strong\u003e\u003c\/h3\u003e\n\u003cp\u003eThere is a tailing phenomenon during electrophoresis. \u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003ePossible reasons:\u003c\/strong\u003e\u003c\/p\u003e\n\u003cp\u003e①Contaminated by RNase during experimental operation; \u003c\/p\u003e\n\u003cp\u003e②Contaminated DNA template by RNase.\u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuggestions: \u003c\/strong\u003e\u003c\/p\u003e\n\u003cp\u003e①Use RNase-free pipette tips and EP tubes, wear disposable latex gloves and masks, and all reagents are prepared with RNase free H2O. \u003c\/p\u003e\n\u003cp\u003e②Re-purify the template DNA.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"top_tabc\" data-v-444b0c7e=\"\"\u003e\n\u003cspan class=\"boxTap\" id=\"tabCBox3\" data-v-444b0c7e=\"\"\u003eCitations \u0026amp; References:\u003c\/span\u003e\n\u003cdiv data-v-444b0c7e=\"\"\u003e\n\u003cdiv data-v-444b0c7e=\"\"\u003e\n\u003cp\u003e\u003cspan\u003e[1] Dong Z, Zheng N, Hu C, et al. Nosema bombycis microRNA-like RNA 8 (Nb-milR8) Increases Fungal Pathogenicity by Modulating BmPEX16 Gene Expression in Its Host, Bombyx mori. Microbiol Spectr. 2021;9(2):e0104821. doi:10.1128\/Spectrum.01048-21(IF:7.171)\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003e[2] Wang X, Tang S, Ye S, et al. Ultrasensitive quantitation of circulating miR-195-5p with triple strand displacement amplification cascade. Talanta. 2022;242:123300. doi:10.1016\/j.talanta.2022.123300(IF:6.057)\u003c\/span\u003e\u003c\/p\u003e\n\u003ch3\u003e\u003cstrong\u003eFAQ\u003c\/strong\u003e\u003c\/h3\u003e\n\u003cp\u003eQ: What is the length of the Control DNA Template?\u003c\/p\u003e\n\u003cp\u003eA: The size is 1142 base pairs.\u003c\/p\u003e\n\u003cp\u003eQ: Does the T7 kit have a cap structure and polyA?\u003c\/p\u003e\n\u003cp\u003eA: The polyA tail is introduced during the design of the template, while the addition of the cap is done separately.\u003c\/p\u003e\n\u003cp\u003eQ: How can the IVT system be optimized?\u003c\/p\u003e\n\u003cp\u003eA: ① Increase the input ratio of NTP and its analogues; ② Reasonable application of additives; ③ The reaction temperature can be set at 40 degrees.\u003c\/p\u003e\n\u003cp\u003eThe optimization of the IVT system can be achieved by adjusting the template, the amount of T7 enzyme used, and if it is co-transcription with cap addition, the ratio between the cap and NTP can be considered; for enzymatic cap addition, the conditions can be optimized in terms of the dosage of the cap-adding enzyme, 2-O-methyltransferase, and SAM. Regarding the template, apart from the requirement of complete linearization, there is another point, which is sequence design. For example, in the 5'UTR region, pay attention to uAUG and Kozak sequence; for CDS, consider the codon preference, GC3 content, and dinucleotide preference, etc.; 3'UTR: RBP and miRNA binding sites: and another point is the secondary structure of RNA. The stable secondary structure of the 5'UTR pre-sequence will reduce the expression of mRNA; the stable secondary structure of 3'UTR will increase the stability of mRNA and promote the expression of the encoded protein. For the recommended IVT system and more detailed tips, please refer to the mRNA in vitro synthesis experiment manual of Vivian Biotechnology.\u003c\/p\u003e\n\u003cp\u003eQ: When the IVT reaction is scaled up from a 20 ul system to a 10 ml system, the yield decreases, and the yield per unit volume of the product drops by approximately 50%. Have you encountered this situation before? Do you have any suggestions?\u003c\/p\u003e\n\u003cp\u003eA: When scaling up the 20uL system proportionally to 10mL, the yield should be approximately the same. Usually, we directly scale up from 20uL to 50mL, and the yield is generally at the level of 100ug\/20uL. For the 50mL volume, we also use centrifuge tubes and a metal bath for measurement. For the 10mL volume, the amplification effect is generally better. In such cases, it is generally recommended to fine-tune the final concentrations of the enzyme and NTP before trying again.\u003c\/p\u003e\n\u003cp\u003eQ: How much of the mRNA integrity can be achieved at present?\u003c\/p\u003e\n\u003cp\u003eA: Regarding the re-submissions from our other clients, based on the CE results measured using Qsep100, the ideal situation is that the CE integrity can reach 88% to 92%; of course, achieving this level usually requires a series of optimizations.\u003c\/p\u003e\n\u003cp\u003eThe average yield is between 120-220 ug\/ul (the yield is highly dependent on the sequence design). In the co-transcription method, the mRNA yield is 8 g\/L. With process optimization, the yield will increase. Currently, the quality parameters of the available processes include observing the fluorescence value of GFP and measuring the cap addition rate using LC-MS.\u003c\/p\u003e\n\u003cp\u003eQ: During your own testing process, do you use agarose gel electrophoresis to detect the mRNA products?\u003c\/p\u003e\n\u003cp\u003eA: We can also conduct RNA electrophoresis. The main purpose is to briefly assess the purity of the products and check for any obvious aggregates, etc. If we are looking at the size of the RNA, during the electrophoresis process, we usually add a sample of a clearly defined RNA size as a reference.\u003c\/p\u003e\n\u003cp\u003eQ: Will you conduct a test for the remaining Rnase in the DNA template?\u003c\/p\u003e\n\u003cp\u003eA: Currently, the detection of the template mainly includes items such as sequence integrity, endotoxin, and residual host nucleic acids. There is no clear detection method for RNase yet; or you can refer to the detection method for residual RNase in our enzyme products, which is relatively simple and reliable.\u003c\/p\u003e\n\u003cp\u003eQ: What is the mechanism for the generation of dsRNA during the IVT reaction?\u003c\/p\u003e\n\u003cp\u003eA: There are several possible reasons for the generation of ds during the IVT process, mainly:\u003c\/p\u003e\n\u003cp\u003eThe T7 pol enzyme will use the product RNA as a template and continue to extend the RNA at its 3' end. The extended sequence will form complementary pairing with the 3' end sequence of the original RNA molecule, thereby creating a hairpin structure.\u003c\/p\u003e\n\u003cp\u003e② The T7 pol enzyme can use RNA as a template to replicate RNA molecules, and synthesize the complementary strand.\u003c\/p\u003e\n\u003cp\u003e(At present, there is no effective method for removing dsRNA. However, there is a patent report suggesting that a fiber column (sigma) could be tried.)\u003c\/p\u003e\n\u003cp\u003eQ: Is there a feasible method to control the content of dsRNA in the products of in vitro transcription?\u003c\/p\u003e\n\u003cp\u003eA: The suggestions regarding optimizing the IVT system and adjusting the reaction conditions to inhibit dsRNA mainly include the following points:\u003c\/p\u003e\n\u003cp\u003eReduce the concentration of magnesium ions; however, reducing the concentration of magnesium ions will also lead to a decrease in the production of RNA, and a balance point needs to be reached.\u003c\/p\u003e\n\u003cp\u003e② Increase the reaction temperature of IVT to 50-56℃. This temperature can effectively reduce the content of dsRNA in the product; however, the requirements for equipment for high-temperature reactions will vary, and it may also increase the cost.\u003c\/p\u003e\n\u003cp\u003e③ In the IVT reaction system, certain dispersants such as urea and formamide are added; our process research team has also conducted some experiments on this route, which can achieve the effect of inhibiting dsRNA. However, whether it is suitable for process scaling-up still needs further research.\u003c\/p\u003e\n\u003cp\u003eQ: What are the reasons for the formation of precipitates during the IVT reaction process, and how can it be optimized?\u003c\/p\u003e\n\u003cp\u003eA: Internal research has studied this precipitation. It is a polymer composed of magnesium ions, RNA, sodium ions, and magnesium pyrophosphate. There is a coagulation effect, and the aggregates keep growing in size (similar gel-like, flocculent, or particulate precipitations all follow a similar principle). The effects of adding water and adding EDTA are similar, which also supports this statement. Reasons: 1. If the concentration of Mg ions is too high, it will aggregate with pyrophosphate to form magnesium pyrophosphate precipitation. However, it has a coagulation effect, continuously encapsulating nucleic acids to form a gel-like substance, resulting in a decrease in yield. 2. A high RNA production level will also cause precipitation.\u003c\/p\u003e\n\u003cp\u003eThis kind of precipitation occurs by chance and is also closely related to the sequence design.\u003c\/p\u003e\n\u003cp\u003eSolution: (1) Reduce the concentration of magnesium ions, but this will result in a decrease in yield. The amount of T7 enzyme needs to be increased and the reaction time (from 2 hours to 3-4 hours) needs to be extended; (2) The form of NTP (the presence of magnesium ions at a high concentration, such as 46 mM, does not select NTP, but when the magnesium ion concentration decreases, the yield of NTP (Tris salt form) does not change much, and the yield of the triNa form of NTP decreases significantly.\u003c\/p\u003e\n\u003cp\u003eQ: What are the considerations for reactor selection during the process scale-up of mRNA production?\u003c\/p\u003e\n\u003cp\u003eA: You could consider WAVE, Haodao Fu reaction vessels, and Sartorius. The main thing is to find bags that are compatible with the existing IVT system. For the stirring tank, the rotational speed of the propeller also needs to be optimized based on the actual situation, as it is related to the type and size of the blades. It is recommended to consult the equipment manufacturer.\u003c\/p\u003e\n\u003cp\u003eQ: Do you recommend the two-step method or the one-step method? Why are most of them still using the two-step method at present?\u003c\/p\u003e\n\u003cp\u003eA: In industry, the most commonly used method for enzymatic cap addition is to treat IVT products with vaccinia virus cap-adding enzyme, which can modify them into Cap 0 mRNA. The Cap 0 structure can be further modified into Cap 1 (m7GpppmN) under the action of 2'O-methyltransferase. With enzymatic cap addition, the cap addition efficiency can reach over 95%. The co-transcription cap addition method is simple to operate, but because GTP competes with the cap dimer, the cap addition rate of this method is a little lower; both methods have their own advantages and disadvantages.\u003c\/p\u003e\n\u003cp\u003eQ: What is the output of IVT in vitro transcription?\u003c\/p\u003e\n\u003cp\u003eA: Currently, our system uses a 20-ul reaction volume. The output of in vitro transcription is 100-200 ug. The internal amplification system with a 10 ml volume can reach 50 mg - 100 mg.\u003c\/p\u003e\n\u003cp\u003eQ: When the reaction time exceeds 2 hours during in vitro transcription, will the completeness and yield decrease? How can the generated mRNA be ensured to be stable?\u003c\/p\u003e\n\u003cp\u003eA: It is recommended that the entire reaction time be between 2 and 3 hours. Our company has verified a maximum reaction time of 3 hours, and our cooperative clients have also verified a reaction time of 4 hours, and the results were normal.\u003c\/p\u003e\n\u003cp\u003eQ: Could you please assist in designing the DNA template sequence for in vitro mRNA transcription (mainly for the application in COVID-19 vaccines)?\u003c\/p\u003e\n\u003cp\u003eA: No related services are provided. Currently, the main reference for the COVID-19 vaccine is still the publicly available vaccine sequences from external sources, namely BioNtech and Moderna. The main original sequences are generally reliable, except for the incomplete polyA tail; the earlier COVID-19 vaccines developed in China are mostly based on the RBD protein sequences. We haven't obtained the specific sequences for this part; basically, these are the core technologies of each research team.\u003c\/p\u003e\n\u003cp\u003eQ: What methods and instruments are used for the detection of the cap rate and tailing effect?\u003c\/p\u003e\n\u003cp\u003eA: The capillary column used for adding the cap is from the Agilent series (6230B TOF), while the tailing column used is from Thermo's QE or Waters' Rda.\u003c\/p\u003e\n\u003cp\u003eQ: Do you use tail-adding enzymes when adding the A tail? How many base pairs do you generally recommend for the connection?\u003c\/p\u003e\n\u003cp\u003eA: This reagent kit does not contain the component for poly A tail. In the template preparation stage, the A tail can be attached to the plasmid, and the length is usually between 20 and 200.\u003c\/p\u003e\n\u003cp\u003eQ: In the product purification process, what is the maximum speed for centrifugation? What is the corresponding rotational speed?\u003c\/p\u003e\n\u003cp\u003eA: Any power rating above 13000 rpm is acceptable.\u003c\/p\u003e\n\u003cp\u003eQ: Why did this B component of the Buffer form a precipitate? How should it be dealt with?\u003c\/p\u003e\n\u003cp class=\"MsoNormal\"\u003eA: The precipitate is spermidine. This is a normal phenomenon and it can be dissolved by hand.\u003cspan\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003ch3\u003e\u003cstrong\u003eRelated blog：\u003c\/strong\u003e\u003c\/h3\u003e\n\u003cp class=\"Polaris-Text--root Polaris-Text--headingLg Polaris-Text--semibold Polaris-Text--base\" tabindex=\"-1\"\u003e\u003ca href=\"\/blogs\/enzyme\/dnase-i-and-their-applications\"\u003e\u003cstrong\u003eDNase I Applications\u003c\/strong\u003e\u003c\/a\u003e\u003c\/p\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n                          \u003c\/div\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n                  \u003c\/div\u003e","brand":"Yeasen Biotechnology","offers":[{"title":"10 T","offer_id":53331732726125,"sku":"10623ES10","price":35.0,"currency_code":"USD","in_stock":true},{"title":"50 T","offer_id":53332190789997,"sku":"10623ES50","price":165.0,"currency_code":"USD","in_stock":true},{"title":"100 T","offer_id":53332190822765,"sku":"10623ES60","price":295.0,"currency_code":"USD","in_stock":true}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/0949\/7424\/7277\/files\/8909-167029822101711082.png?v=1778795252","url":"https:\/\/www.ebiohippo.com\/products\/t7-high-yield-rna-synthesis-kit-bht20800006","provider":"BioHippo","version":"1.0","type":"link"}