Quantum prep plasmid miniprep kit

To molecularly characterize the identified virus species, amplification products obtained from 12 samples (Table 1) selected among those resulted positives for HpMV (2), HpLV (9) and AHLV (1), were cloned and sequenced.
Amplicons were cleaned by Amicon Ultra-0.5 Centrifugal Filter Units (Merck, Boston, MA, USA), ligated into the pGem-T vector and cloned according to the manual instruction of the pGem-T-Easy vector system (Promega, Madison, WI, USA). Transformed plasmids were isolated by Quantum Prep^® Plasmid Miniprep Kit (Bio-Rad, Hercules, CA, USA) and submitted to Sanger sequencing on both ends (Eurofins Genomics, Konstanz, Germany) employing the Sp6 and T7 primers. The obtained sequences were analysed and compared using the web software Clustal Omega (EMBL-EBI) https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 30 August 2023) and Nucleotide BLAST available online at the web page https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 30 August 2023). Nucleotide sequences of the ORF5 coding for the putative CP were then included to phylogenetic analysis (Maximum Likelihood method based on the Kimura 2-parameter model, with a bootstrap phylogenetic test (1000 replicates) performed using MEGA X software [35 (link)].

Total DNA extraction was performed using the Wizard^® genomic DNA purification kit (Promega Cat. No. A1120). Plasmid DNA was isolated using the Quantum Prep Plasmid Miniprep Kit (Biorad Cat. No. 732-6100). For DNA digestion, the manufacturer’s instructions were followed (Roche and New England Biolabs). Separated DNA fragments were recovered from agarose using the high pure PCR cleanup micro kit (Roche Cat No. 04983955001). Alkaline phosphatase, ligation reactions, Southern blots and colony hybridization were performed by standard protocols (Sambrook et al., 1989 ). DNA digoxigenin-dUTP probes were obtained via PCR following the instructions of the manufacturer (Roche). The Expand high fidelity PCR system (Roche Cat. No. 11732641001) was used for the amplification of PCR fragments for cloning. Sequences of these PCR fragments were verified in order to discard amplicons containing mutations. Competent cells were prepared using calcium chloride and transformations were performed by standard protocols (Sambrook et al., 1989 ). Highly electrocompetent cells were prepared as previously reported (Choi et al., 2006 (link)) and transformed using an EC100 electroporator according to the manufacturer’s instructions (EC apparatus corporation).

To molecularly characterize the identified virus species, amplification products obtained from 12 samples (Table 1) selected among those resulted positives for HpMV (2), HpLV (9) and AHLV (1), were cloned and sequenced.
Amplicons were cleaned by Amicon Ultra-0.5 Centrifugal Filter Units (Merck, Boston, MA, USA), ligated into the pGem-T vector and cloned according to the manual instruction of the pGem-T-Easy vector system (Promega, Madison, WI, USA). Transformed plasmids were isolated by Quantum Prep^® Plasmid Miniprep Kit (Bio-Rad, Hercules, CA, USA) and submitted to Sanger sequencing on both ends (Eurofins Genomics, Konstanz, Germany) employing the Sp6 and T7 primers. The obtained sequences were analysed and compared using the web software Clustal Omega (EMBL-EBI) https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 30 August 2023) and Nucleotide BLAST available online at the web page https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 30 August 2023). Nucleotide sequences of the ORF5 coding for the putative CP were then included to phylogenetic analysis (Maximum Likelihood method based on the Kimura 2-parameter model, with a bootstrap phylogenetic test (1000 replicates) performed using MEGA X software [35 (link)].