Hello,
We would like to in vitro generate mRNA from this plasmid, encoding SB100X transposase https://www.addgene.org/browse/sequence/178440/ using mMessage mMachine T7 Transcription Kit. We don't have experience with plasmids yet, so we would like to ask, which is the best restriction site to cut the plasmid in order to linearize it before in vitro transcription. Should we cut with MfeI or SalI or any of the other sites is preferrable?
Thank you very much in advance
Hey Anny,
our freshly puplished paper describes excatly that topic. It describes linearisation of plasmids (where to cut, how primers are designed and so on) and in vitro mRNA transcription with the linearized plasmids with a T7-Promotor. I would be happy, if you would take a look at:
Herb, M., Farid, A., Gluschko, A., Krönke, M., Schramm, M. Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA. J. Vis. Exp. (153), e60143, doi:10.3791/60143 (2019).
If you have any further questions, please ask.
All the best,
Marc
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