Categories
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Which dose should I use for my in vitro and in vivo experiments?
As the dose is cell-type-dependent, the dose you use for one cell-type might not give you equal results in another. Therefore, we advise to test and try at least 3 doses of mRNA and determine linearity for the specific cell type you are using. A safe range to stay in for a well-designed mRNA is around 50-70 ng/96-well, although some cells will tolerate up to 200-250 ng per 96-well. At RIBOPRO, we often use 10, 50, and 100 ng to determine the optimal dose. Keep in mind that the dose scales (linearly) with a larger surface of cells.
For in vivo use, the species and the target organ make a huge difference in the recommended dosing. For mice, we generally recommend staying below 1-2ug/25gram animal.
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Can I use my own plasmid for the mRNA synthesis?
Yes, we would be happy to use your DNA plasmids for the synthesis of your mRNA sequence. 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)
• A 5′ and 3′ UTR sequence
• Linearization site after 3’ UTR. Preferably a type IIS restriction site creating a 5’ overhang.
• Kozak site (GCCACC(ATG)If you are unsure whether your plasmid meets the requirements, feel free to contact one of our Product Specialists, they are happy to help!
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How should I store my mRNA?
At -20°C the mRNA is stable for approximately one month, while at –80°C the mRNA is stable for at least a year. We advise to aliquot the mRNA sample the first time you thaw the mRNA, so that the efficacy and stability of the product does not decrease by repeated freeze-thaw cycles.
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What is the minimum and maximum length of custom mRNA sequences?
We can synthesize custom mRNA sequences starting from 100nt up to 15k nt.