Trans2k plus 2 dna marker

The in vitro transcripts were initially purified by undergoing gel purification on a 5% urea-PAGE gel (200 V at room temperature for 1 h). Following this, in vitro RNA folding was performed according to previously described methods [19 (link)]. Briefly, the in vitro transcripts were first dissolved in 0.5× TE buffer at pH 8.0. They were then subjected to heating at 95 °C for 3 min, followed by cooling on ice for 5 min. Subsequently, the RNA was incubated at 37 °C for 5 min in a folding buffer consisting of 500 mM Tris–HCl at pH 7.5 and 500 mM NaCl. After adding MgCl₂ to a final concentration of 10 mM, the mixture was further incubated for 30 min. Electrophoresis was then performed using 5% native PAGE gel (80 V for 4 h at 4 °C), with each sample containing 50 ng of RNA. For comparison, PSTVd-C was included as a control [21 (link)]. Trans2K^® Plus II DNA Marker (Transgen Biotech, Peking, China) was also loaded as a size marker. Images were captured after ethidium bromide staining under UV light.

Phusion DNA polymerase (Thermo Fisher, USA) was used to amplify DNA fragments. If the size of the DNA fragment was longer than 10 kb, PrimeSTAR GXL DNA polymerase (TaKaRa, Japan) was used. The Trans 2K Plus II DNA marker (TransGen Biotech, Beijing) was used as a DNA ladder to measure the size of DNA fragments by agarose gel electrophoresis. A gel extraction kit, plasmid extraction minikit, and BAC/PAC (P1-derived artificial chromosome) DNA isolation kit (all from Omega, USA) were used to purify DNA. All primers were synthesized by the Beijing Genomics Institute. Magnesium chloride, manganese chloride, rubidium chloride, polyethylene glycol 8000 (PEG 8000), PEG 3350, and DMSO were purchased from Sigma-Aldrich (USA), and the remaining reagents were purchased from Sangon Biotech (Beijing, China).