Ribomax large scale rna production systems kit

The ZIKV-Nanoluciferase (Nanoluc) construct (Fig. 1A) used in these assays was described previously^{34 (link)}. For maintenance and propagation of the plasmid containing the pCCI-SP6-ZIKV-Nanoluc, the E. coli Turbo strain (New England Biolabs) was used.
Complete amplification of the viral genome was performed using a PCR reaction with Phusion High Fidelity (Thermo Fisher) enzyme and the designed primers ZIKV-Forward (5′ CG ATT AAG TTG GGT AAC GCC AGG GT 3′) and ZIKV-Reverse (5′ T AGA CCC ATG GAT TTC CCC ACA CC 3′). The PCR product containing SP6 promoter followed by complete viral cDNA was purified with the DNA clean and concentration kit (Zymo Research). In vitro transcription was performed using the RiboMAX™ Large-scale RNA Production Systems kit (Promega), as instructed by the manufacturers.

To make the single guide RNA (sgRNA), we first PCR amplified a dsDNA template, which contained the consensus sequence from a DNA plasmid (pgRNA-bacteria plasmid from Addgene), using a sgRNA FWD primer that contained a T7 promoter, and sgRNA REV primer. The following thermal cycling conditions were used to generate the PCR template: 98 °C for 3 minutes; 98 °C for 10 seconds; 65 °C for 20 seconds; 72 °C for 15 seconds; go to 98 °C for 10 seconds; 65 °C for 20 seconds; 72 °C for 15 seconds for 29 cycles and 72 °C for 8 minutes. The PCR template was verified using gel electrophoresis (1.5% agarose, 1× TBE buffer, 120 V for 90 minutes) and subsequently purified using the WizardSV Gel and PCR Clean-Up System (Promega) according to the manufacturer's instructions. sgRNA was then transcribed from the PCR template using the RiboMax™ Large Scale RNA Production Systems kit (Promega) according to the manufacturer's instructions. Following transcription, RNA products were purified using the RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's instructions. RNA quality was verified using gel electrophoresis (Mini-Protean TBE-Urea Precast Gels (Bio-Rad), 200 V for 30 minutes). Gels were visualized under UV light in a Biorad ChemiDOCT MP imaging system.