Rnase free turbo dnasei

TYLCV infected and control plants were monitored along 21 days. The second most recently expanded leaf from the apex was harvested at 2, 7, 14 and 21 days-post infection (dpi). The presence of viral DNA was analysed in each test plant at 21 dpi by hybridization of tissue blots^{64 (link)}. For each infection method, time point and replica, the leaf tissue from 6 infected plants was pooled and used in downstream analysis. A total of three biological replicates were processed per condition and time point.
Total DNA was isolated with the DNeasy Plant Mini Kit (Qiagen). Total RNA was isolated using the Trizol reagent (Ambion), and 3 μg were set aside for construction and sequencing of small RNAs libraries. Total RNA was treated with RNase free Turbo DNaseI (Ambion) according to the manufacturer’s guidelines and cleaned up by a phenol:chloroform treatment.
Absolute quantification of the virion-sense (VS) and the complementary-sense (CS) strands was performed following the protocol described by⁵² with the following modifications: 15 ng of total DNA were used for the VS and CS strand synthesis step and 2 μl of a 1:2 dilution of the purified DNA, were used as the template for the qPCR. Total viral DNA was quantified by standard qPCR using 3 ng of total DNA.

Total RNA was isolated from A. hydrophila AH-3, AH-3::flrA, AH-3::flrBC, AH-3::fliA_p, AH-3::lafK, and AH-3::lafS which were grown at 25°C in liquid media (TSB) or plates (TSA) by RNA Protect Bacteria Reagent (Qiagen) and RNeasy Mini kit (Qiagen). To ensure that RNA was devoid of contaminating DNA, the preparation was treated with RNase-free TurboDNase I (Ambion). First-strand cDNA synthesis was carried out using the Thermoscript RT-PCR system (Invitrogen) and random primers on 5 μg of total RNA DNase-digested, according to the manufacturer’s instructions. PCR without reverse transcriptase was also performed to confirm the absence of contaminating DNA in the RNA sample. The second strand synthesis and subsequent DNA amplification of flgT internal fragment was carried out using the BioTaqDNA polymerase (Bioline) and the pair of oligonucleotides5′-CAGTGGCTGG ACGAGAAC-3′ and 5′- TTCCAATACTGCCAGATCC-3′ designed using the Prime program from the Genetics Computer Group package (Madison, Wisconsin). Amplicons were visualized by agarose gel electrophoresis with ethidium bromide staining. A. hydrophila ribosomal 16S primers were used as a control of cDNA template.