Zymo rna clean and concentrator 25 kit

All mRNA generating plasmids were digested with PvuII-HF (NEB R[Δ4]151 L) and ApaLI-HF (NEB R0507L) at 37 °C for 3 h. After digestion, templates were run on a 1% agarose gel to confirm cutting and the reactions were purified with Zymo DNA Clean and Concentrator-5 (Zymo Research D4013). 1ug of linearized template was used in a 100 uL T7 RNA Polymerase (purified in house) reaction that was incubated at 37 °C for 3 h. After T7 reactions were complete, 15uL TurboDNAse (ThermoFisher Scientific AM2238) was added, and reactions were incubated at 37 °C for 15 min. The RNA was then purified using a Zymo RNA Clean and Concentrator-25 Kit (Zymo Research R1017). Eluted RNA was measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific Q32852) then capped following the protocol for the Vaccinia Capping System (NEB M2080S) and purified one last time using Zymo RNA Clean and Concentrator-5 (Zymo Research R1013). Capped RNA concentrations were measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific Q32852). Final RNA was diluted to 0.25pmoles/ul for use in the in vitro translation assays.
For the comparing capped versus uncapped mRNA, uncapped RNA was incubated for 5 min at 65 °C to match the treatment of capped RNAs. The same RNA that was used in the vaccinia capping reaction was directly compared to the post-cap RNA.