Considerations when using kits for mRNA production

• Consider restriction enzyme cut site design early in project for both cost and design components; e.g., some REs are contraindicated for use due to generation of 3' overhang which can compromise downstream IVT applications due to generation of extraneous transcripts.
• Maintain RNase-free conditions in your RNA lab space to include use of appropriate cleaning and handling protocols, and full segregation of any bacterial growth or purification events (especially relevant if individuals are also generating their own linear pDNA). This also includes QC space and equipment.
• Bring all cold components to room temperature before assembling the IVT reaction to avoid precipitating the DNA template.
• If your template encodes a poly(A) tail, use recombinant-deficient hosts such as recA, endA type strains for plasmid propagation.
• Avoid exceeding 25% of the volume of the growth vessel to ensure maximum aeration.
• Consider yield variability that may occur when generating larger constructs (> 5 kb) or reactions utilizing modified NTPs or cap analogs.
• Consider yield variability and purification attrition that may occur when scaling reactions down to very small quantities (20 - 50 µL).
• Store RNA long term at -80C to maintain stability.

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