I use the MEGAscript T7 Transcription Kit (Cat. no.: AMB13345, ThermoFisher) for in vitro transcription of linearized plasmids. However, I have measured a high concentration of DNA in my mRNA after in vitro transcription. I use 15-20 ng of plasmid for the reaction, and I have already incorporated a second DNase treatment step in my protocol, but it doesn't seem to remove the DNA. I use DeNovix for measurement of concentrations. Anyone who has experience with this? If yes, is your mRNA still functional even though DNA is present?
It should be fine. Just know that if you didn't do a cleanup step to remove the DNAase etc. then the DNA is still present (just chopped into pieces). What are you planning to do with the transcripts?
Hi Amy! Thank you for your answer :)
I use the RNA Clean & Concentratior^TM- kit (R1018, ZYMO RESEARCH) for RNA clean-up. Do you have experience with that? If yes, is that sufficient enough to remove DNA and DNases?
I will use the transcripts to develop an mRNA vaccine
It will not be an issue. You can run a UREA PAGE gel to separate DNA and RNA bands, cut your RNA band, and purify to get pure RNA by electroelution or crush and soak.
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