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.
