T7 RNA Synthesis Kit med højt udbytte -10623.
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T7 RNA Synthesis Kit med højt udbytte -10623.
T7 RNA Synthesis Kit med højt udbytte -10623.
T7 High Yield RNA Synthesis Kit optimerer transkriptionsreaktionssystemet. Sættet kan syntetisere det enkeltstrengede RNA effektivt ved at bruge T7 RNA-polymerase, det lineære dobbeltstrengede DNA med T7-promotorsekvensen som template, NTP'er som substrat til at kontrollere DNA-sekvensen nedstrøms for promotoren. Under transkription kan modificerede nukleotider tilføjes til substratet for at fremstille biotin eller farvestof-mærket RNA. Dette kit kan syntetisere lange transkripter og korte transkripter, RNA kan produceres 100-200 μg med 1 μg DNA skabelon input. RNA'et syntetiseret ved transkription kan bruges til forskellige downstream-applikationer, såsom RNA-struktur- og funktionsforskning, RNase-beskyttelse, probehybridisering, RNAi, mikroinjektion...

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T7 RNA Synthesis Kit med højt udbytte -10623.

YeasenSKU: 10623ES10
Størrelse: 10 t
Pris:
$35.00
Lager:
På lager

T7 High Yield RNA Synthesis Kit optimerer transkriptionsreaktionssystemet. Sættet kan syntetisere det enkeltstrengede RNA effektivt ved at bruge T7 RNA-polymerase, det lineære dobbeltstrengede DNA med T7-promotorsekvensen som template, NTP'er som substrat til at kontrollere DNA-sekvensen nedstrøms for promotoren. Under transkription kan modificerede nukleotider tilføjes til substratet for at fremstille biotin eller farvestof-mærket RNA.

Dette kit kan syntetisere lange transkripter og korte transkripter, RNA kan produceres 100-200 μg med 1 μg DNA skabelon input. RNA'et syntetiseret ved transkription kan bruges til forskellige downstream-applikationer, såsom RNA-struktur- og funktionsforskning, RNase-beskyttelse, probehybridisering, RNAi, mikroinjektion og in vitro oversættelse.

Figur 1: In vitro RNA-transkriptionsproces

Feature

  • Op til 180 μg RNA pr. reaktion fra 1 μg af kontrolskabelonen
  • Optimeret reaktionssystem til IVT-processen
  • Reducer dsRNA-produktionen
  • Højere RNA-integritet og renhed

Anvendelse

  • In vitro RNA syntese

Komponenter

Komponenter nr. Navn 10623ES50 (50 T) 10623ES60 (100 T) 10623ES70 (500 T)
10623-A T7 RNA polymerase blanding 100 μL 200 μL 1 ml
10623-B 10×transskriptionsbuffer 100 μL 200 μL 1 ml
10623-C ATP (100mM) 100 μL 200 μL 1 ml
10623-D CTP (100 mm) 100 μL 200 μL 1 ml
10623-E GTP (100 mm) 100 μL 200 μL 1 ml
10623-F UTP (100 mm) 100 μL 200 μL 1 ml
10623-G Kontrol DNA skabelon (500ng/μL) 10 μL 20 μL 100 μL

Forsendelse og opbevaring

Tøris transport. Opbevares ved -15℃ ~ -25℃, gyldig i to år.

Figurer

Figur 1. Standard-RNA blev syntetiseret in vitro under anvendelse af T7 RNA-syntesesæt.

Reaktionen blev inkuberet i et PCR-instrument ved 37°C i 2 timer og derefter oprenset med magnetiske perler (kat #12602). Udbytteresultatet blev analyseret med NanoDrop spektrofotometer som vist i figur 1.

Figurer 2. Transskriptionsdemonstrationen af ​​forskellige længder af RNA ved hjælp af T7-kit i henholdsvis elektroforetogrammet (figur 2A), kapillærelektroforesediagrammet (figur 2B) og kromatogrammet (figur 2C)

Figur 3. Syntese af capped RNA in vitro.

Reaktionen blev inkuberet i PCR-instrument ved 37 ℃ i 2 timer og derefter oprenset med magnetiske perler (kat #12602). Udbytteresultatet blev analyseret med NanoDrop spektrofotometer som vist i figur 3A. Integritetsresultatet blev analyseret ved kapillarelektroforese som vist i figur 3B.

Dokumenter:
Ofte stillede spørgsmål:

1. Lavt afskriftsudbytte

Kvaliteten af ​​skabelonen er tæt forbundet med udbyttet. Hvis udbyttet af forsøgsgruppen er væsentligt lavere end kontrolgruppen, er de mulige årsager:

① den eksperimentelle skabelon indeholder hæmmende komponenter;

② Skabelonen har noget galt.

Forslag:

① Rens skabelonen igen;

② Bestem skabelonkvantificeringen og dens integritet;

③ Forlæng reaktionstiden;

④ Øg mængden af ​​skabeloninput;

⑤ Prøv andre promotorer og RNA-polymeraser.

2. Lavt udbytte af korte afskrifter

Et kort transkriptionsinitieringsfragment vil hæmme reaktionen. Når transkriptionsproduktet er mindre end 100 nt, vil forlængelse af reaktionstiden til 4-8 timer eller øge mængden af ​​skabelon til 2 μg øge RNA-udbyttet.

3. RNA-transkriptionslængden er større end forventet

Hvis elektroforesen viser, at produktbåndet er større end den forventede størrelse, er de mulige årsager:

① Plasmidskabelonen er muligvis ikke fuldstændig lineariseret;

②3'-enden af ​​sense-strengen har en fremtrædende struktur;

③ RNA'et har en sekundær struktur, der ikke er fuldstændig denatureret.

Forslag:

①Tjek om skabelonen er fuldstændig lineariseret, og udfør om nødvendigt yderligere linearisering;

②Vælg et passende restriktionsenzym for at undgå 3'-overhæng, eller brug Klenow Fragment/T4 DNA-polymerase til at fuldføre transkriptionen, før du fortsætter;

③Brug denatureret gel til at detektere RNA-produkter.

4. RNA-transkriptionslængden er mindre end forventet

Hvis elektroforesen viser, at produktbåndet er mindre end den forventede størrelse, er de mulige årsager:

①Skabelonen indeholder en termineringssekvens svarende til T7 RNA-polymerase;

②GC-indholdet i skabelonen er højt.

Forslag:

①Sænk reaktionstemperaturen (for eksempel 30°C). Nogle gange kan en sænkning af temperaturen øge transskriptionslængden, men det vil reducere udbyttet. Eller prøv forskellige RNA-polymeraser til transkription;

②Hvis skabelonens GC-indhold er højt, skal du bruge 42℃ til transskription, eller tilføj SSB for at øge udbyttet og transskriptionslængden.

5. Elektroforese-tailing af transkriptionsprodukter

Der er et halefænomen under elektroforese.

Mulige årsager:

① Kontamineret med RNase under eksperimentel drift;

② Kontamineret DNA skabelon af RNase.

Forslag:

①Brug RNase-fri pipettespidser og EP-rør, brug latex-engangshandsker og -masker, og alle reagenser er forberedt med RNase-fri H2O.

②Genrens skabelon-DNA'et.

Citater og referencer:

[1] Dong Z, Zheng N, Hu C, et al.Nosema bombycis mikroRNA-lignende RNA 8 (Nb-milR8) øger svampepatogenicitet ved at modulere BmPEX16-genekspression i dens vært, Bombyx mori. Microbiol Spectr. 2021;9(2):e0104821. doi:10.1128/Spectrum.01048-21(IF:7.171)

[2] Wang X, Tang S, Ye S, et al. Ultrafølsom kvantificering af cirkulerende miR-195-5p med triple-streng displacement amplification kaskade. Talanta. 2022;242:123300. doi:10.1016/j.talanta.2022.123300(IF:6.057)

Storage

Dry ice transportation. Store at -15℃ ~ -25℃, valid for two years.

FAQ

The size is 1142 base pairs.
The polyA tail is introduced during the design of the template, while the addition of the cap is done separately.
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.
The 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.
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.
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.
The 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.
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.
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.
There are several possible reasons for the generation of ds during the IVT process, mainly:
The 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.
The T7 pol enzyme can use RNA as a template to replicate RNA molecules, and synthesize the complementary strand.
(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.)
The suggestions regarding optimizing the IVT system and adjusting the reaction conditions to inhibit dsRNA mainly include the following points:
1. Reduce 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.
2. 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.
3. 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.
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.
This kind of precipitation occurs by chance and is also closely related to the sequence design.
Solution: (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.
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.
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.
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.
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.
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.
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.
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.
Any power rating above 13000 rpm is acceptable.
The precipitate is spermidine. This is a normal phenomenon and it can be dissolved by hand.

Citations

Documents

Files
MSDS-10623-A-T7_RNA_Polymerase_Mix-HB221014.pdf
MSDS-10623-B-10x_Transcription_Buffer-HB221014.pdf
MSDS-10623-C-ATP_100_mM_-HB221014.pdf
MSDS-10623-D-CTP_100_mM_-HB221014.pdf
MSDS-10623-E-GTP_100_mM_-HB221014.pdf
MSDS-10623-F-UTP_100_mM_-HB221014.pdf
MSDS-10623-G-DNA_template-HB221014.pdf

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