Author: Martin, Baptiste; Coutard, Bruno; Guez, Théo; Paesen, Guido C; Canard, Bruno; Debart, Françoise; Vasseur, Jean-Jacques; Grimes, Jonathan M; Decroly, Etienne
Title: The methyltransferase domain of the Sudan ebolavirus L protein specifically targets internal adenosines of RNA substrates, in addition to the cap structure Document date: 2018_9_6
ID: 243u68j8_7
Snippet: Codon-optimized SUDV MTase+CTD synthetic genes (Biomers) were cloned into a pET14b vector for expression in bacteria. Mutations were introduced by PCR-amplifying the wild-type sequence using primers carrying the mutations, using the Turbo DNase (Ambion). PCR-products were purified using the Wizard SV PCR Clean-Up System (Promega). Transformed Escherichia coli T7 bacteria (NEB) were cultured at 30 • C until an O.D. of 0.6 was reached, after whic.....
Document: Codon-optimized SUDV MTase+CTD synthetic genes (Biomers) were cloned into a pET14b vector for expression in bacteria. Mutations were introduced by PCR-amplifying the wild-type sequence using primers carrying the mutations, using the Turbo DNase (Ambion). PCR-products were purified using the Wizard SV PCR Clean-Up System (Promega). Transformed Escherichia coli T7 bacteria (NEB) were cultured at 30 • C until an O.D. of 0.6 was reached, after which the temperature was changed to 17 • C and IPTG (Sigma) was added to 20 M. The next day, bacteria were spun down (8000 × g for 10 min at 4 • C) using a Sorval Lynx 6000 centrifuge (Thermo), and pellets were stored at -80 • C.
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