Purelink quick gel extraction and pcr purification combo kit

100 µl of PCR reaction of each amplified product belonging to neurotoxin biosynthetic genes (anatoxin-a and saxitoxins) was purified using the PureLink™ Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer protocol followed by direct sequencing (Macrogen, Madrid Spain). The obtained nucleotide sequences were initially examined using BLAST and the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/BLAST).

The PCR‐templates were purified by PureLink® Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen) following the producer's protocol and quantified using the Qubit dsDNA broad‐range assay (Invitrogen). The amplicons from genomic DNA were Sanger sequenced by Eurofins Scientific (Ebersberg, Germany). The amplicons from eDNA‐samples were pair‐end sequenced (2 × 250 bp) with the Illumina MiSeq platform by Norwegian Sequencing Centre (Oslo, Norway).

The products obtained from all molecular assays were purified with a commercial kit (PureLink Quick Gel Extraction and PCR Purification Combo Kit; Invitrogen Life Technologies, Carlsbad, CA, USA), quantified using a Qubit Fluorometer (Invitrogen Life Technologies, Eugene, OR, USA), and then submitted for direct sequencing in both directions with the forward and reverse primers used in the respective molecular assays. Sequencing was performed using an ABI3500 Genetic Analyzer sequencer with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems^®, Foster City, CA, USA).
Sequence quality analyses and consensus sequences were obtained using the PHRED and CAP3 webpage (http://asparagin.cenargen.embrapa.br/phph, accessed on 8 January 2024), respectively. The identities of the nucleotide (nt) sequences obtained were compared with the nt sequences deposited in GenBank via BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 8 January 2024) analysis. Selected sequences were deposited in GenBank.

All bacterial transformants were checked for identity by PCR to prevent contamination. Total DNA was extracted from GFP-tagged bacteria of three different constructs from overnight culture by using a MasterPure Complete DNA and RNA purification kit (Epicentre, Madison, WI, USA) according to the manufacturer's protocol. The bacterial 16S rRNA genes were amplified using universal primers, 27f (5′- AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′). PCR was performed in a final volume of 50 μl using 10 μM of each primer, 10 mM concentration of deoxynucleoside triphosphates, 50 mM MgCl₂, 1 U of Taq polymerase and buffer (Invitrogen, Carlsbad, CA, USA). Denaturation was performed at 95°C for 2 min, followed by 30 cycles of 95°C for 30 s, annealing at 54°C for 30 s, and 72°C at 1 min 30 s. The final extension was at 72°C for 7 min. PCR products were purified using the PureLink Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Carlsbad, CA, USA). The purified PCR products were sent for Sanger sequencing. DNA sequences were assembled with DNA baser sequence assembly software (http://www.dnabaser.com). The assembled sequences were used for blast searches at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).

The products of the molecular assays designed to amplify the nucleic acids of OvGHV2, BoGHV6, and BCoV from the DNS, lung, and intestine of these animals were purified using the PureLink Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen Life Technologies, Carlsbad, CA, USA), quantified by using a Qubit Fluorometer (Invitrogen Life Technologies, Eugene, OR, USA), and submitted to direct sequencing in both directions with the forward and reverse primers used in the respective molecular assays in an ABI3500 Genetic Analyzer sequencer with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).
Sequence quality analyses and consensus sequences were obtained using PHRED and CAP3 software (http://asparagin.cenargen.embrapa.br/phph/), respectively. Similarity searches of the OvGHV2 tegument protein, the BoGHV-6 polymerase, and the BCoV N genes were performed with the respective nucleotide (nt) sequences deposited in GenBank using the Basic Local Alignment Search Tool software (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The identity of the nt sequences was confirmed by comparison with reference sequences available in GenBank.

A full length MTAP-ANRIL cDNA was ligated with linearized PCR 2.1-TOPO vector (TOPO TA cloning, Invitrogen, USA). Plasmid DNA was used as a template and the insert sequence was re-amplified using forward primer with NdeI restriction site (Fwd-primer_NdeI, Supplementary Table S3) and M13 as reverse primer. The amplicon was gel purified (PureLink Quick Gel Extraction and PCR Purification Combo Kit, Invitrogen, USA). The gel purified product and circular pET20b(+) vector were digested overnight with respective restriction enzymes (NdeI and EcoRI in MaMel95; NdeI and BamHI in MaMel30). Following the inactivation of the enzymes and column purification (MinElute Reaction Cleanup Kit, Qiagen, Germany), the digested product was incubated overnight at 16°C using T4 ligase (T4 DNA Ligase, New England Biolabs, Germany). The ligation mix was transformed into DH-5α (Z-Competent Cells, HiSS Diagnostics Germany) and plasmid DNA sequenced to confirm the position and orientation of the cloned fragment.