Product Number: TR03

Shipping and Storage

Store at -20°C℃±5℃.

Component

Description

As a biomacromolecule, mRNA can be synthesized at scale through in vitro transcription (IVT). The T7 promoter is currently one of the most efficient promoters for transcription, enabling rapid and straightforward production of large quantities of RNA molecules using T7 RNA polymerase. As a byproduct of IVT, double-stranded RNA (dsRNA) can be recognized by corresponding nucleic acid receptors, triggering innate immune inflammatory responses that severely impact the efficacy of mRNA vaccines. Strict control during production is essential. This product employs a T7 RNA polymerase engineered through molecular evolution, significantly reducing dsRNA levels in transcription products and lowering the immunogenicity of synthesized mRNA.

This product is a GMP-grade recombinant T7 RNA polymerase produced through large-scale fermentation using Escherichia coli. It is manufactured with pharmaceutical-grade raw materials and excipients, with strict control over host protein residues, nucleic acid residues, and other impurities. The production and quality management processes comply with GMP standards, ensuring traceability of the entire production process and all raw materials.

Application

1. Synthesize single stranded RNA for the preparation of mRNA vaccines and other applications.
2. Synthesize highly specific RNA probes. 
3. Synthesize siRNA precursor.
4. Preparation of RNA splicing precursors.
5. Synthesize capped RNA using cap analogues.

Quality control

Storage buffer solution

50mM Trizma base; 100mM NaCl; 1mM EDTA; 20mM β-ME; 50% (v/v) Glycerol; 0.1% Triton X-100; pH 7.5.

Source

E.coli carrying bacteriophage T7 RNA polymerase gene

Features

It exhibits high specificity for the T7 promoter.

Definition of active units

The enzyme amount required to incorporate 1nmol of [³H] GMP into an acid insoluble precipitate within 1 hour at 37℃ and pH 8.0 is defined as 1 active unit.

Related Products

TR01 – T7 RNA Polymerase

TR02 – Thermostable T7 RNA Polymerase

TR03 – T7 RNA Polymerase 200U/ul

GMP-T701 – T7 RNA Polymerase, GMP Grade

E131 – T7 High Yield RNA Transcription kit

Description

S-adenosylmethionine is the most important methyl donor in vivo and an auxiliary substrate involved in methyl transfer reaction. In the synthesis of mRNA in vitro,both the hydroxyl group of ribose and the amino group of base need the participation of SAM during methylation.

Feature

The SAM is prepared in 0.005M H₂SO₄ and 10% EtOH solution, filtered to provide in the form of aseptic liquid. In the process of mRNA capping, the combination of SAM and methyltransferase can transform the Cap0 structure of the 5′ cap region into Cap1 structure.

Specification

Customized Spesfication:

1）Endonuclease Activity (Nicking) – A 50 µl reaction in Buffer 2 containing 1 µg of supercoiled PhiX174 DNA and a minimum of 5 µl of S-adenosylmethionine (SAM) incubated for 4 hours at 37ºC results in <20%conversion to the nicked form as determined by agarose gel electrophoresis.

2）Non-Specific DNase Activity (16 Hour) – A 50 µl reaction in NEBuffer 2 containing 1 µg of PhiX174-HaeIII DNA and a minimum of 5 µl of S-adenosylmethionine (SAM) incubated for 16 hours at 37ºC results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.

3）Restriction Digest (CpG Resistant, SAM) – A 20 µl reaction in 1X Buffer 2 containing 1 µg of LambdaDNA, 1 unit of M. SssI (CpG Methyltransferase), and 160 µM S-adenosylmethionine (SAM) is incubated for 1hour at 37°C. The resulting DNA is resistant to digestion with BstUI as determined by agarose gel electrophoresis

Order

Product Number: CA341

Shipping and Storage

Store at -20℃±5℃.

Component

Description

As a biomacromolecule, mRNA can be synthesized on a large scale through in vitro transcription (IVT). The T7 promoter is currently one of the most efficient promoters for transcription. Therefore, using T7 RNA polymerase (T7 RNA polymerase) for in vitro transcription can easily and quickly obtain a large number of RNA molecules. This kit optimizes the co transcription reaction system and uses T7 RNA polymerase 2.0, a T7 RNA polymerase mutated enzyme obtained through molecular evolution, which can significantly reduce the production of dsRNA during transcription.

This co transcription kit can use DNA containing T7 promoter sequence, AG promoter sequence, and poly (A) tail coding region as templates, and incorporate CAP1 GAG cap analogs during transcription to obtain mRNA with 5 ‘- Cap1 and 3’ – poly (A) tails. The 5 ‘- Cap1 structure can prevent mRNA from being degraded by nucleases, thereby maintaining mRNA stability and initiating translation, which has a significant impact on mRNA stability, translation efficiency, and immunogenicity in vivo or in cells.

This reagent kit can transcribe 150-250μg of RNA in one reaction, and the co transcription cap rate is greater than 95%. The synthesized RNA can be used for research on RNA structure and function, RNA enzyme protection, probe hybridization, etc RNAi、 Downstream applications such as microinjection and in vitro translation.

Application

Synthesize single stranded RNA with CAP1 GAG structure, which can be replaced with other cap analogs for use.

Product Number: M062011       

Shipping and Storage

Ice pack transportation, stored at -20℃, with a shelf life of 1 year.

Component

Description

This product is a restriction endonuclease type II, derived from a recombinant protein encoded by the BspQI gene in Bacillus sphaericus expressed in E. coli. Its recognition sequence is 5 ‘- GCCTTCN1/N4-3’, mainly used for enzyme digestion of plasmids to prepare poly (A/T/G/C) terminated linearized DNA fragments and obtain specific sticky ends.

Specification

Note: For specific product quality inspection data and more indicators, please refer to the batch quality inspection report.

Unit definition

1 unit: The amount of enzyme required to digest 1μg of λDNA within 1h at 50℃ in a 50μL system.

Product Number: DI05

Shipping and Storage

 -20℃，Valid for 12 months.

Components

Description

DNase I is a non-specific nuclease cleavage enzyme, mostly derived from recombinant E. coli strains, containing MBP fusion clones of bovine pancreatic DNase I. DNase I can be used to degrade single or double stranded DNA, based on the principle that DNase I hydrolyzes phosphate diester bonds to produce single or oligonucleotides with 5 ‘- phosphate groups and 3’ – OH.

Both Mg²⁺ and Mn²⁺ can activate the activity of DNase I, and the concentration of Ca²⁺ directly affects the activity of the enzyme. When Mg²⁺ is present, it can randomly generate incisions on each single strand of double stranded DNA; In the presence of Mn²⁺, double stranded DNA can be broken, resulting in DNA fragmentation.

DNase I solution (1mg/ml) is composed of DNase I, enzyme protection solution, preservatives, etc., with a concentration of 1mg/ml, used for single stranded DNA, double stranded DNA, chromatin, RNA: DNA hybrid strands. It is commonly used for the preparation of DNA free RNA, reverse transcription, and in vitro transcription experiments.

Unit definition

1 unit refers to the amount of enzyme required to completely degrade 1μg of pBR322 DNA in a 50μl reaction system at 37℃ for 10 minutes.

Product Number: GMP-T701            Animal-free          Ampicillin-free

Shipping and Storage

At -20±5℃.

Description

As a biological macromolecule, mRNA can be synthesized on a large scale by in vitro transcription (IVT). T7 promoter is one of the most efficient promoters at present. Therefore, T7 RNA polymerase can be used for in vitro transcription to obtain more synthetic products. T7 RNA polymerase is a T7 promoter-specific, DNA-dependent, 5’→3′ RNA polymerase from T7 bacteriophage. Usingdouble stranded DNA as the template, it transcribes RNA complementary to the single stranded DNA located at the downstream of T7promoter. T7 RNA polymerase has been commonly used for in vitro mRNA synthesis.

The polymerase is GMP Grade produced in E. coli. Our manufacturing processes are strictly controlled to ensure the end products free from host protein or nucleic acid contaminations and other impurities following the Pharmaceutical Manufacturing Guidelines. We guarantee the manufacturing and quality control comply with GMP regulation for tracking each and every step of the manufacturing process, including raw material sourcing.

This product has completed the DMF record of FDA and passed the HALAL certification.

Quality Elements

Annotation: ChP refers to the Pharmacopoeia of the People’s Republic of China.

Complying to following regulations

1. ISO 9001:2015, certified facility.
2. 《GMP Appendix – Cellular therapeutic product》National Medical Products Administration.
3. 《The Pandect of Genetic Therapeutic Product for Human》Chinese Pharmacopoeia Commission.
4. USP Chapter <1043>, Ancillary Materials for Cell, Gene, and Tissue-Engineered Products.
5. USP Chapter <92>, Growth Factors and Cytokines Used in Cell Therapy Manufacturing.
6. Ph. Eur. General Chapter 5.2.12, Raw Materials of Biological Origin for the Production of Cell-based and Gene Therapy Medicinal Products.

Feature

Highly specific for T7 promoter, suitable for RNA in vitro synthesis.

Application

1. Single stranded RNA synthesis
2. RNA probe synthesis.
3. siRNA precursor synthesis
4. Precursor for RNA splicing preparation
5. Capped RNA synthesis.

Examples

Fig: RNA transcription in vitro.

1. From left to right, the quantity of T7 RNA Polymerase copies were 20U, 4U, 0.8U.
2. DNA templates were segments of 2Kb.

Unit definition

At 37℃, pH8.0, within 1 hour, the amount of enzyme required that will incorporate 1nmol tritium labeled GMP into acid-insoluble material is defined as one unit of enzyme activity.

Storage buffer

100mM NaCl; 50mM Tris-HCl (pH 7.9); 1mM EDTA; 20mM 2-mercaptoethanol; 0.1% Triton X-100; 50% (v/v) Glycerol。

Related Products

TR01 – T7 RNA Polymerase

TR02 – Thermostable T7 RNA Polymerase

TR03 – T7 RNA Polymerase 200U/ul

GMP-T701 – T7 RNA Polymerase, GMP Grade

E131 – T7 High Yield RNA Transcription kit

ARCA Ammonium Solution
Product Number: CAP03
Molecular Weight: 817.4 (free acid)
Molecular Formula: C22H32N10O18P3 (free acid)
Appearance: Clear Colorless Solution
Storage Conditions: -20℃
Purity: HPLC≥95%, 100 mM

Product Description

The phosphatase gene isolated from Antarctic psychrophilic strain TAB5 was expressed in Escherichia coli and purified for several times. Phosphatase can catalyze the removal of 5 ´ phosphate groups at the ends of DNA or RNA. After the carrier DNA is treated with thermosensitive phosphatase during cloning, its fragment lacks the 5 ´ phosphate terminal necessary for ligase, so the carrier cannot be self-connected, which can greatly reduce the background of self-connection of the carrier. The enzyme can act on the 5 ´ protruding end, concave end and flat end.

Application

1) Remove the phosphoric acid group at the 5 ´ end of the carrier to reduce the self-connecting background;

2) Remove dNTP and pyrophosphate residues in PCR products;

3) Remove the phosphate group at the 5 ´ end of the DNA probe and prepare the optimal substrate for the 5 ´ end phosphorylation labeling;

4) Dephosphorylation of proteins.

Usage suggestions

Heat sensitive phosphatase at 1 × TSP Buffer has the optimal enzyme activity. If the reaction is to be carried out in the restricted enzyme digestion buffer, 10 must be added to the reaction system × TSP Buffer to 1 × The final concentration was incubated at 37 ℃.

Storage temperature

-20℃。

Package

Quality Control

After several times of column purification, only clear and single target band can be seen in SDS-PAGE gel detection; There was no residual DNA of Escherichia coli and no contamination of endonuclease and exonuclease detected by PCR.

Reaction conditions

1×TSP Reaction Buffer [50 mM Bis Tris propane HCl, 1 mM MgCl2, 0.1 mM ZnCl2 (pH 6.0 at 25 ℃)], incubated at 37 ℃.

Unit definition

At 37 ℃ for 30 minutes, the amount of enzyme required to dephosphorylate 1ug pUC19 DNA Digested by Hind III (producing 5 ´ protruding end), EcoR V (producing smooth end) or Pst I (producing 5 ´ concave end) is defined as a unit.

Dephosphorylation standard refers to inhibit the reconnection of more than 95% DNA fragments in the connection reaction (detected by electrophoresis).

Thermal deactivation

70 ℃ for 5 minutes.

Description

         SP6 RNA polymerase is a DNA dependent RNA polymerase that specifically recognizes the SP6 promoter sequence (5′-ATTTAGGTGACACTATAGAAGNG-3′). SP6 RNA polymerase can catalyze the incorporation of NTP of single stranded or double stranded DNA template downstream of SP6 promoter to synthesize RNA complementary to DNA template downstream of SP6 promoter. Linear plasmids containing SP6 promoters or PCR products containing SP6 promoters can be used as templates for RNA synthesis in vitro.

Accessory reagent

Source

This product is expressed by Escherichia coli, and the expression gene is phage SP6 RNA polymerase gene.

Application

1. Preparation of RNA vaccines and drugs;

2. Synthesis of RNA for in vitro translation;

3. Preparation of radiolabeled RNA probes;

4. Synthesis of antisense RNA for experimental study of expression regulation;

5. Synthesis includes mRNA, siRNA, gRNA and other RNA precursors;

6. Synthesis of Capped mRNA with (Cap analog) as primer.

Storage

Store at -20 ℃ for 24 months.

Storage buffer

50mM Tris-HCl(pH7.9), 100mM NaCl, 1mM EDTA, 20mM β- Mercaptoethanol, 0.1% Triton® X-100, 50% glycerin.

1× RNA polymerase reaction buffer composition

40mM Tris HCl (pH7.9), 10mM DTT, 6mM MgCl2, 2mM spermidine.

Unit Definition

100μL system, one unit enzyme is defined as the amount of enzyme required to add 5 nmol of ATP into polynucleotides within 1 hour at 37 ℃.

Enzyme activity test conditions

50μL reaction system, it contains 1× RNA polymerase reaction buffer, 2mM NTP (0.5mM ATP, GTP, CTP, UTP respectively), 10mM DTT, 1μg DNA template with SP6 promoter.

Quality control

SP6 RNA polymerase does not contain other nucleases, such as T7 or T3 RNA polymerase, DNase and RNA enzyme.

Recommended protocol for RNA synthesis in vitro

1. Template preparation: DNA template containing SP6 promoter and target gene (PCR amplification product, or linearized plasmid). If you use enzyme digestion to linearize the plasmid, it is better to use a restriction endonuclease with a flat end (such as EcoR V), or a sticky terminal enzyme with a protruding 5′ end (such as SapI or BspQI).

2. Precautions before experiment: Make sure that the entire experimental environment is free of RNA enzyme pollution. Use a nozzle, centrifuge tube, and ultrapure water that are free of RNA enzyme. Make sure that the entire reaction system is prepared without RNA enzyme pollution. Make sure that the experimental table, pipette gun, electrophoresis equipment, etc. are free of RNA enzyme pollution. Try to wear masks, hats, and gloves.

3. Reaction system configuration (recommended, system optimization recommended)

4. Incubate at 37 ℃ for 2h.

5. Use DNase I to remove template DNA contamination (optional): every 10μL system add 1U DNase I and incubate at 37 ℃ for 30min. DNase I can be inactivated by heating at 65 ℃ for 10min.

Reference

1. Simon Stammen, Franziska Schuller, Sylvia Dietrich, Martin Gamer, Rebekka Biedendieck, Dieter Jahnl (2010). Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivoprotein production in Bacillus megaterium. Appl Microbiol Biotechnol (2010) 88:529–539.

2. W.Tom Stump, Kathleen B. Hall(1993), SP6 RNA polymerase efficiently synthesizes RNA from short double-stranded DNA templates, Nucleic Acids Research, 5480-5484 Nucleic Acids Research, 1993, Vol. 21, No. 23.

3. Jiyun Yoo 1, Changwon Kang (2000), Bacteriophage SP6 RNA polymerase mutants with altered termination efficiency and elongation processivity, Genetic Analysis: Biomolecular Engineering 16 (2000) 191–197.

4. E D Jorgensen, R K Durbin, S S Risman, W T McAllister (1991), Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters, Volume 12, Issue 7, November 2019, Pages 908-931.

5. Hirokazu Kotaoi, Yukuo Ishizalci, Nobutsugu Hiraoka and AJrira Obayashi(1987),Nucleotide sequence and expression of the cloned gene of bacteriphage SP6 RNA polymerase, Volume 15 Number 6 1987.

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