Takara IVTpro T7 mRNA Synthesis Kit includes a highly efficient T7 RNA polymerase optimized to give high yields of mRNA through in vitro transcription. We offer two product options: Takara IVTpro T7 mRNA Synthesis Kit (Cat. #6144) containing a wild-type T7 RNA Polymerase ver.2.0 (sold separately as Cat. #2541A), and Takara IVTpro T7 mRNA Synthesis Kit (low dsRNA) (Cat. #6134) containing a modified PrimeCap T7 RNA Polymerase (sold separately as Cat. #2560A) for the reduction of double-stranded RNA (dsRNA) generation. All kits allow integration with capping systems (not included) and RNA modification with modified NTPs of your choice.
Once your gene of interest is seamlessly cloned into the template vector, linearize the plasmid with a restriction enzyme such as HindIII or BspQ I (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, verify that the IVT template 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 tenfold. 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 the final desired concentration (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 (8 mM). 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)
