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). 

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.

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). 

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.

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).

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