Uv gel doc xr system

DsRNA was extracted from fungal mycelium and purified virus particles, according to the method of Valverde et al. [32 ] as modified by Khalifa and Pearson [33 (link)]. For visualization the dsRNA was mixed 5:1 with loading dye (30% glycerol, 1 X tris-borate-EDTA-buffer (TBE), 2% ficoll-400, 0.25% xylene cyanol, 0.25% bromophenol blue) and the mix loaded into a 1% agarose gel prepared in 0.5X TBE buffer and pre-stained with 5 μl RedSafe nucleic acid stain (Intron) per 100 mL. The gel was run in 0.5 X TBE buffer at 90 V for ~ 45 min and visualised using a UV Gel Doc XR+ system (BIO RAD). The size of the dsRNA bands was estimated against a 1 kb plus DNA ladder (Invitrogen). The dsRNA nature of the bands was confirmed as described by Howitt et al. [34 ].

To overcome restriction modification and improve the transformation efficiency of CRISPRi (pJT3) plasmids into V. parahaemolyticus, we treated pJT3 plasmids with V. parahaemolyticus cell extract to methylate plasmid DNA. Plasmids were isolated from E. coli using the Qiagen Plasmid Midi Kit (Cat#12,145) following the manufacturer’s instructions. First, 100 μg pJT3 plasmids were incubated with 100 μL (containing ca. 350 μg V. parahaemolyticus cell extract protein) in methylation reaction buffer (20 mM Tris–acetate (pH 7.9), 50 mM potassium acetate, 5 mM Na₂EDTA, 1 mM DTT and 200 mM S-adenosyl-L-methionine) at 37 °C for 30 min. Then, following the manufacturer’s instructions, the plasmid DNA was purified with a Qiagen mini-preparation kit (Cat#12,125).
After methylation and DNA purification, the plasmid was subjected to restriction enzyme SalI (NEB, cat#R3138S) digestion, and reaction mixtures were analyzed by 1% agarose gel electrophoresis. The gel was then stained with ethidium bromide (Merck, Cat#E8751) and imaged on a UV Gel Doc XR + System (Bio-Rad).