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  • ‹ Back to Takara IVTpro mRNA Synthesis System
  • Cloning Kit for mRNA Template
  • Takara IVTpro T7 mRNA Synthesis Kit
Home › Learning centers › mRNA and cDNA synthesis › mRNA synthesis › Takara IVTpro mRNA Synthesis System › Takara IVTpro T7 mRNA Synthesis Kit

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Product overview

Takara IVTpro T7 mRNA Synthesis Kit

Takara IVTpro T7 mRNA Synthesis Kit (Cat. # 6144) includes a highly efficient T7 RNA polymerase optimized to give high yields of mRNA through in vitro transcription. The kit also allows integration with capping systems (not included) and RNA modification with modified NTPs of your choice. 

Components
10X Transcription Buffer
10X ATP
10X CTP
10X GTP
10X UTP
10X Enzyme Mix
Nuclease-Free Water
DNase I
Lithium Chloride Precipitation Solution
Positive Control Template (FLuc) (0.5 µg/µl)
Template linearization Co-transcriptional capping Incorporation of modified rNTPs Positive Control Template (FLuc) Reagents not included in this kit

Template linearization  

Once your gene of interest is seamlessly cloned into the template vector, linearize the plasmid with a restriction enzyme such as HindIII (not included) to create the double-stranded DNA containing the T7 promoter sequence. The template linearization ensures IVT transcripts are produced at a uniform length.

Takara IVTpro T7 mRNA Synthesis Kit is optimized for use with cap analogs such as CleanCap Reagent AG (TriLink Biotechnologies, Cat. # N-7113, not included). Please see the Co-transcriptional capping section for details. If using any IVT template vector other than the Linearized Template Vector in the Cloning Kit for mRNA Template (Cat. # 6143), verify that it has a transcription start site compatible with the cap analogs used in the IVT reaction.

The IVT reaction run with the Takara IVTpro T7 mRNA Synthesis Kit can be scaled up 10-fold. A consistent mRNA yield across this range has been demonstrated using CleanCap Reagent (Figure 1). 

Figure 1. The relationship between IVT reaction volume (20–200 μl) and total mRNA yield is consistent. Positive Control Template (FLuc) was used along with CleanCap Reagent AG (3’ OMe).

Takara IVTpro T7 mRNA Synthesis Kit also contains DNase I to digest the template DNA after the IVT reaction and a lithium chloride (LiCl) solution to purify the synthesized mRNA.

Co-transcriptional capping  

Eukaryotic translation requires a cap structure at the 5′ end of RNA. Takara IVTpro T7 mRNA Synthesis Kit is optimized for use with CleanCap Reagent AG to generate mRNA with a cap structure. We recommend using CleanCap Reagent AG or CleanCap Reagent AG (3’ OMe) at a 4:5 molar ratio with NTP for a final concentration of 8 mM (Table 1). 

Reagent Volume
Nuclease-Free Water X μl
10X Transcription Buffer 2 μl
10X ATP 2 μl
10X CTP 2 μl
10X GTP 2 μl
10X UTP 2 μl
CleanCap Reagent AG* 1.6 μl
Template DNA* 1 μg
10X Enzyme Mix 2 μl
TOTAL 20 μl

*Component not included in the kit.

Table 1. An IVT reaction using CleanCap Reagent AG is shown. This example is also found in the User Manual for Takara IVTpro T7 mRNA Synthesis Kit and Takara IVTpro mRNA Synthesis System.

Incorporation of modified rNTPs  

Ribonucleoside triphosphates (rNTPs) in Takara IVTpro T7 mRNA Synthesis Kit are provided in a separate tube so that modified NTP(s) can be easily incorporated. Studies show that using pseudo UTP (not included) reduces the immunogenicity of the host cells (Kariko et al., 2008). When using a modified NTP, replace the corresponding NTP provided in the kit with an equivalent amount in the IVT reaction.

Positive Control Template (FLuc)  

Pre-linearized FLuc Positive Control Template (4,574 bp) can be used as assay control for in vitro transcription reactions. The positive control template has a sequence composed of T7 promoter + 5'-UTR + FLuc-CDS + 3'-UTR + Poly(A) (Figure 2). Download sequence information for the Positive Control Template (fasta format).

Figure 2. Positive Control Template (FLuc). The vector is pre-linearized by the HindIII restriction site as indicated.

Reagents not included in this kit  

  • CleanCap Reagent AG (TriLink BioTechnologies, No. N-7113 or N-7413)
  • Modified nucleotides such as pseudo-UTP
  • Restriction enzyme (HindIII etc.)

Related Products

Cat. # Product Size License Quantity Details
6141 Takara IVTpro™ mRNA Synthesis System 1 Set USD $398.00

Takara IVTpro mRNA Synthesis System includes all components necessary from DNA template preparation to high-yield in vitro transcription reaction. Easily scale up to 10-fold (200 μl reaction volume) without affecting the expected mRNA yield. This kit includes the Cloning Kit for mRNA Template (Cat. # 6143) and Takara IVTpro mRNA Synthesis Kit (Cat. # 6144), which are also sold separately.

Notice to purchaser

This product is for in vitro diagnostic use. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from Takara Bio Inc. If you require licenses for other use, please contact us by phone at +81 77 .565. 6976 or from our website at https://www.takara-bio.co.jp/inquiry/ivd/. Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. All trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions.

Documents Components Image Data

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Affect of CleanCap Reagent AG (3’ OMe) on RNA yield and the expression of FLuc mRNA

Affect of CleanCap Reagent AG (3’ OMe) on RNA yield and the expression of FLuc mRNA

Addition of the CleanCap Reagent AG (3’ OMe) in IVT reaction has no significant effect on mRNA yield (Panel A). HEK293T cells transfected with mRNA containing a cap structure show significatly higher protein expression compared to cells transfected with mRNA without a cap (Panel B).

Back

Increasing IVT reaction volume has no effect on RNA yield or quality.

Increasing IVT reaction volume has no effect on RNA yield or quality.

The mRNA yield was proportional to the reaction volume up to 200 µl with no significant change in mRNA concentration (Panel A). Scaling up of the IVT reaction volume does not affect the quality of RNA product (Panel B).

Back

Effect of different RNA purification methods on RNA yield and protein expression.

Effect of different RNA purification methods on RNA yield and protein expression.

Purification of RNA product using LiCl was comparable to purification with NucleoSpin column when two elutions were performed (Panel A). HEK293T cells transfected with mRNA purified using LiCl shows protein expression comparable to mRNA purified using NucleoSpin columns (Panel B).

Back

6141: Takara IVTpro mRNA Synthesis System

6141: Takara IVTpro mRNA Synthesis System
6143 Cloning Kit for mRNA Template 10 Rxns USD $179.00

Cloning Kit for mRNA Template enables easy cloning of a template DNA into a pre-linearized plasmid (included in the kit) for downstream in vitro transcription reactions. The pre-linearized plasmid contains the T7 promoter, transcription initiation sequence (AGG), 5'- and 3'-UTRs (untranslated region), and poly(A) sequence. The kit also includes an In-Fusion Snap Assembly Master Mix for seamless cloning of the target DNA into the pre-linearized vector and a FLuc Control Fragment, consisting of 15 bp 3' and 5' In-Fusion sequences and Fluc CDS (1,683 bp).

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

6143: Cloning Kit for mRNA Template

6143: Cloning Kit for mRNA Template
6144 Takara IVTpro™ T7 mRNA Synthesis Kit 20 Rxns USD $248.00

Takara IVTpro mRNA Synthesis Kit enables the production of large amounts of mRNA through in vitro transcription using an optimized, highly efficient T7 RNA polymerase. The kit includes separate NTPs for easy optimization or replacement with modified nucleosides, such as pseudouridine. The kit synthesizes up to 200 μg or more of mRNA per 20 μl reaction, includes DNase I to digest the template DNA after the in vitro transcription reaction and a lithium chloride (LiCl) solution to purify the synthesized mRNA for downstream applications. 

The kit also includes a Positive Control Template (FLuc) plasmid containing the T7 promoter, transcription initiation sequence (AGG), 5'- and 3'-UTRs, FLuc CDS, and poly(A) sequence.

Easily scale up to 10-fold (200 μl reaction volume) without affecting the expected mRNA yield.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

Affect of CleanCap Reagent AG (3’ OMe) on RNA yield and the expression of FLuc mRNA

Affect of CleanCap Reagent AG (3’ OMe) on RNA yield and the expression of FLuc mRNA

Addition of the CleanCap Reagent AG (3’ OMe) in IVT reaction has no significant effect on mRNA yield (Panel A). HEK293T cells transfected with mRNA containing a cap structure show significatly higher protein expression compared to cells transfected with mRNA without a cap (Panel B).

Back

Increasing IVT reaction volume has no effect on RNA yield or quality.

Increasing IVT reaction volume has no effect on RNA yield or quality.

The mRNA yield was proportional to the reaction volume up to 200 µl with no significant change in mRNA concentration (Panel A). Scaling up of the IVT reaction volume does not affect the quality of RNA product (Panel B).

Back

Effect of different RNA purification methods on RNA yield and protein expression.

Effect of different RNA purification methods on RNA yield and protein expression.

Purification of RNA product using LiCl was comparable to purification with NucleoSpin column when two elutions were performed (Panel A). HEK293T cells transfected with mRNA purified using LiCl shows protein expression comparable to mRNA purified using NucleoSpin columns (Panel B).

Back

6144: Takara IVTpro T7 mRNA Synthesis Kit

6144: Takara IVTpro T7 mRNA Synthesis Kit

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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