Dear colleagues,

I made some mRNA product of 910 bp, from a restriction-enzyme-linearised pUC-derived vector. Standard NEB IVT T7 cleancap synthesis kit protocol was followed (E2080 NEB) as 2x20uL reactions with substitution of UTP to 100% N1-methylpseudouridine. Reactions were incubated at 37'C for 3 hours, held at 4'C in thermocycler overnight. mRNA samples were then topped up with nuclease-free water to 50 uL and 2-uL DNAse1 was added and incubated for 15 minutes at 37C. Standard NEB T2040 50-ug purification protocol was followed and mRNA was eluted into 50 uL of nuclease-free water. Concentrations were checked on Implen. Instead of getting a usual yield of around 2000-3000 ng/uL, I only got ~25 ng/uL which is not typical. None of the protocols were changed. The only thing that was changed is the insert product in the vector. I cannot think of any reason, apart of RNAse contamination, either in reagents or during the procedure. mRNA was synthesised in biosafety hood with RNAse-free items and the workspace and pipettes were decontaminated with RNAseZap and UVed for 20 minutes before worrking with RNA.

Running the sample on Agilent tapestation gave strange result and I am not sure how to interpret it. Instead of 910 bp band, I got ~250 bp bands and it does not look like an RNAse contamination, but there is a lot of strange, low-weight product. Any suggestions?

Thanks,

Kind regards,

Maria