Transcript aidtm t7 high yield transcription kit

The variant PLMVd.282 was dimerized and cloned as previously described [31] (link). The RNA was transcribed from a SpeI-digested plasmid using the TranscriptAidTM T7 High Yield Transcription Kit (Fermentas) and following the manufacturer’s instructions in order to generate the (+) polarity strand. The RNA was then purified by phenol:chloroform extraction followed by ethanol precipitation. The resulting RNA pellets were washed in 70% ethanol and then dissolved in loading buffer (95% formamide, 10 mM EDTA, pH 8.0, 0.025% xylene cyanol and 0.025% bromophenol blue). The samples were then fractionated through 5% denaturing polyacrylamide gels (PAGE, 19∶1 ratio of acrylamide to bisacrylamide) in buffer containing 45 mM Tris-borate, pH 7.5, 8 M urea and 2 mM EDTA. The dimeric RNA strand was extracted from the gel during a 16 h elution in an elution buffer containing 500 mM ammonium acetate, 10 mM EDTA and 0.1% SDS. After elution, the RNA was ethanol precipitated, washed with 70% ethanol, dried and dissolved in ultrapure water.
A healthy GF-305 peach tree obtained from a peach seedling was grown in greenhouse conditions prior to its slash inoculation with the dimeric RNA of PLMVd.282 dissolved in 50 mM KH₂PO₄.

The potentially regulatory roles of Mn-SDHB on male sexual development in M. nipponense were analyzed by using RNAi. Snap Dragon tools,⁴ which were used to design the specific RNAi primer with T7 promoter site, shown in Table 2. The Transcript Aid^TM T7 High Yield Transcription kit (Fermentas, Inc, United States) was used to synthesize the Mn-SDHB dsRNA, following the procedures of the manufacturer. A total of 300 healthy mature male M. nipponense were collected with body weights of 3.12–4.87 g, and divided into two groups. As described in previous study (Jiang et al., 2014 ; Li et al., 2018 (link)), the prawns from experimental group were injected with 4 μg/g Mn-SDHB dsRNA. The concentration of the Mn-SDHB dsRNA was adjusted to 4 μg/μl. Thus, the injected volume of Mn-SDHB dsRNA into each prawn was the same as that of their body weight, while the prawns from the control group were injected with equal volume of vehicles based on the body weight. The SDHB mRNA expression was investigated in the testis by qPCR after the injection of 1, 7, and 14 days, in order to detect the interference efficiency (N ≥ 5).

The M. tuberculosis ssrA (tmRNA) gene under the control of the T7 promoter was amplified by PCR from M. tuberculosis H37Rv genomic DNA with primers M containing the T7 promoter sequence and N (Table 4). The M. tuberculosis tmRNA was transcribed in vitro and purified according to the manufacturer’s protocol of the Transcript Aid^TM T7 High Yield Transcription Kit (Fermentas).