We would be happy to use your DNA plasmids for the synthesis of your mRNA sequence as this may save time and money.
For us to utilize your plasmid in the synthesis of high-quality mRNA, your plasmid should meet the following requirements:
• T7 promoter upstream of the 5′ UTR
• T7 promoter should preferably be followed by three G nucleotides ….TATA(GGG) in case you desire enzymatic capping.
• For capping by means of trinucleotide cap analogues, we require T7 promoter immediately followed by AG, as in ….TATA(AG)…, or followed by AU, as in ….TATA(AU)… depending on the variant of the cap analogue.
• A 5′ and 3′ UTR sequence flanking the gene of interest, optionally including a translation initiation site, such as Kozak, in the 5’UTR
• Optionally an encoded poly(A) tail of no longer than 120nt (please note that we cannot guarantee exact poly(A) length)
• Linearization site after 3’ UTR. Preferably a type IIS restriction site creating a 5’ overhang (Please check if the enzyme cuts at the correct position of the template strand (often the bottom strand reading 5′ to 3′ from the T7 promoter)).
Other requirements:
• Name: Please inform us of the name of the restriction enzyme to be used.
• Format: Circular plasmid. Please do not perform linearization yourself as residual restriction enzyme may cause issues in our process.
• Quality: single band on agarose gel of correct size. In particular, avoid nicked pDNA, which shows up on gel as secondary (higher running) bands.
• Quantity: Approximately 7 µg of plasmid for 1 mg of RNA production.
• Vector backbone: Provide the name and map. Please inform us on GMO classification. (Does the coding DNA sequence encode a harmful gene product or contain the complete genome of a viral pathogen classified as risk group 4 ,3 or 2?*)
• Insert Gene: Is it encoding a toxic or viral protein?
Plasmid Handling Guidelines:
• If possible, supply the plasmid lyophilized.
• If lyophilization is not feasible, please provide in RNase-free water at a concentration of 100 ng/µL.
• Ensure the plasmid is high-purity and endotoxin-free
• Please label the tube with the plasmid and insert names.
If you are unsure whether your plasmid meets the requirements, feel free to contact us.
