Thermo scientific genejet pcr purification kit

The PCR products which were amplified in nested PCR using the primer set GNAS-379F1+GNAS-1040R1 were purified using the Thermo Scientific GeneJET PCR purification Kit (Fisher Scientific, Oslo, Norway) and direct sequencing was performed using the light run sequencing service of GATC Biotech (https://www.gatc-biotech.com/en/index.html). The BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) program was used for computer analysis of sequence data against the reference sequence with accession number NM_000516.4.

Bisulfite sequencing was used to determine the percentage methylation at individual CpG sites within the Art3, Pcdhb6, Rsp16, Tspo, and Wnt16 promoters. Details of PCR conditions and primers are detailed in Table 2. Briefly, 1 µl of diluted bisulphite-treated DNA was added as a template in a PCR reaction using 10 µl Hot Star Taq mastermix (Qiagen), 1 µl of forward, and reverse primers (at a concentration of 10 pmol/µl) in a total volume of 20 µl. Amplification was carried out in a Mastercycler Nexus Gradient thermocycler (Eppendorf) using the following protocol; 95°C for 15 min, then 40 cycles of 95°C for 30 s, annealing temperature for 1 min, 72°C for 1 min, followed by 72°C for 5 min. Three microlitres of PCR product was used for visualization on a 1% agarose-TAE gel subjected to electrophoresis to confirm the presence of a PCR product. The remaining 17 µl PCR products were subsequently purified for sequencing using Thermo Scientific GeneJET™ PCR Purification Kit (Fisher Scientific, UK) to remove primers, dNTPs, and other impurities from the PCR product, according to the manufacturers’ instructions.

RT-PCR products of Lamon bean samples that tested positive for the presence of BCMV (23 samples), PSV (12 samples), and CMV (7 samples) were sequenced through Sanger technology. Amplicons were purified using a Thermo Scientific GeneJET PCR Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol and sequenced through Sanger technology by BMR Genomics (http://www.bmr-genomics.it/ accessed 7 November 2020). Nucleotide sequences were analysed with Blast sequence analysis tools [64 (link)].
To compare with previous phylogenetic works in the literature, reference sequences of strains detected in this investigation were aligned and analyzed with those included in the most recent phylogenetic studies on BCMV [27 (link)], CMV [28 (link)] and PSV [29 (link)]. Phylogeny of BCMV, PSV and CMV isolates infecting Lamon bean plants was reconstructed by adopting the maximum likelihood (ML) method as implemented in Geneious—R10 (Biomatters Ltd., Auckland, New Zealand). To test the robustness of each branch, a total of 500 bootstrap replicates were performed. Sanger sequences were also aligned to corresponding HTS contigs with Blastn [64 (link)] to obtain alignment identity values.