Qiamp dneasy blood and tissue kit

DNA from filtered urine samples were extracted by QIAmp DNeasy^® Blood and Tissue Kit (Qiagen, Hilden, Germany). The filter paper quadrant used for draining the urine was used to punch 15 holes (~1mm diameter) by regular paper punch. The paper punch and scissor was sterilized by washing with 10% bleach and nuclease-free-water after every use for minimizing any chance of contamination [9 ]. The paper discs were added to nuclease-free-water in 2 ml Eppendorf tubes. These were then heat shocked at 95°C for 10 minutes. After heat shock, the tubes were left on a shaker at 150 rpm for overnight.
The next morning, tubes were removed from the shaker and centrifuged at 8,000 x g for 1 minute. The liquid in tube was transferred into a QIAmp 2 ml column tube. Then DNA was extracted according to the manufacturer’s protocol. All extracted DNA was quantified by using Nanodrop (Thermo Scientific, Wilmington, DE, USA) to determine the concentration of the extracted DNA after which all the extracts were stored at -20°C until use.

Genomic DNA was extracted using the QIAmp DNeasy blood and Tissue Kit (Qiagen) following manufacturer instructions. Extracted DNA was stored at 4 °C for Polymerase Chain Reaction (PCR) amplification. A fragment of mitochondrial cytochrome C oxidase subunit 1 (cox1) (~381 bp) was amplified using the conserved forward primers: 5′-TTTTTGGGCATCCTGAGGTTTAT-3′ and reverse primers: 5′-TAAAGAAAGAACATAATGAAAATG-3′ [44 (link)], under the following conditions: initial denaturation at 94 °C for 5 min, followed by 35 cycles of 30 s denaturation at 94 °C, 30 s annealing at 55 °C, and 72 °C for 30 s, followed by a final extension at 72 °C for 7 min. The PCR reaction mixture contained: 25 µL Master Mix (GoTaq1 Green Master Mix, Promega, Madison, WI, USA), which contained 1 U Taq polymerase, 400 µM dNTP, 3 mM MgCl₂, and 0.3 µM of each primer, 5 μL of DNA template, and nuclease-free water was added up to 50 µL PCR reaction mixture. A positive and negative control (without genomic DNA) was amplified in each PCR reaction. PCR products (10 μL) were run on 1.5% agarose gel electrophoresis to detect the single bands. After purification by the QIAquick Gel Extraction Kit (Qiagen), the PCR products were sent for sequencing to Biomed, Beijing, China.

Total DNA was extracted from 477 fin clips in a clean room using QIAmp DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) and following the manufacturer's protocol. Partial fragments of the mitochondrial (mt) cytochrome c oxidase subunit 1 (COI) gene (the so‐called barcoding region) and the mt cytochrome b (cytb) gene were amplified using the primer pairs FishF1/FishR1 (COI; Behrens‐Chapuis et al., 2015 ) and GluF/ThrR (cytb; Bergsten et al., 2014 ) with the polymerase chain reaction (PCR) protocol described in (Palandačić et al., 2017 (link)). Sequencing was performed at Microsynth (Balgach, Switzerland) in both directions using the PCR primers.
Sequences were manually checked, trimmed and unambiguously aligned (no missing data) in Geneious v2.10.3 (https://www.geneious.com). COI and cytb sequences were subsequently concatenated using Geneious, and the concatenated alignment, including available sequences from GenBank, was used for all further genetic analysis (hereinafter referred to as combined COI–cytb).

DNase assays were performed as per our published protocol (Chaurasia et al., 2022a (link)). Briefly, the reaction was set up with purified HeLa cell genomic DNA (QiAmp DNeasy Blood and Tissue kit; Qiagen, Invitrogen, United States), in a final volume of 15 μL 1X (TM) sample buffer comprising 10 mM Tris and 3 mM MgCl₂ (pH 7.5). Recombinant VM proteins at a concentration of 30 nM were equilibrated in TM buffer to 22 °C prior to use and later 150 ng of genomic HeLa DNA was added, and the reaction was terminated at 30 min upon addition of premixed gel loading dye (New England Biolabs, United States). Gels were stained with ethidium bromide (0.5 μl/ml) and samples were analyzed by 1% agarose gel electrophoresis. The gel images were taken using Gel Doc UV illumination (Gel Logic 212 Pro, Carestream Molecular imaging, United States). The docking study was performed for the C-terminal region of LA3490 with a phosphate or magnesium ion using MGLTools 1.5.7. The size of grid box was set to 17.34 Å × 5.782 Å x 8.06 Å at x-, y-, and z-axis coordinates respectively. Lamarckian genetic algorithm was used as scoring function. Genetic algorithm and local search algorithm were responsible for the search of possible confirmation degree of freedom of ligands and protein.