Invitrogen sybr safe dna gel stain

To identify CPV-2-positive samples, PCR amplification of VP2 was conducted using the conventional method. For each PCR reaction, 25 µL of DreamTaqTM PCR Master Mix (2×) (Thermo Fisher Scientific^®) was used, with 4 µL of the Forward Ext1F primer (5′-ATGAGTGATGGAGCAGTTCA-3′) and 4 µL of the Ext3R Reverse primer (5′-AGGTGCTAGTTGAGATTTTTCATATAC-3′) described by [17 (link)] and 17 µL of a mixture of DNA and molecular grade water, reaching an amount of 500 ng DNA for each reaction. Molecular grade water was used as a negative control for amplifications. PCR protocol used was as follows: an initial denaturation cycle at 94 °C for 5 min, 35 denaturation cycles at 94 °C for 30 s, alignment at 50 °C for 45 s, extension at 72 °C for 1 min and a final extension cycle at 72 °C for 5 min.
PCR amplification results were visualised using 1.5% horizontal agarose gel electrophoresis. Gels were stained with the Invitrogen ™ SYBR® Safe DNA Gel Stain (Thermo Fisher Scientific^®). In each well, 4.2 μL of each sample obtained after amplification and 0.8 μL of the 6× DNA loading buffer were used, and the GeneRuler™ 100-bp DNA Plus Ladder (Thermo Fisher Scientific^®) was used as a molecular weight marker. Gels were developed in the ultraviolet light Gel Doc™ XR+ imaging system (Bio-Rad, Molecular imager^®, USA) and were visualised using ImageLab™ software.

DNA was extracted by standard protocols, including several purification steps for eliminating humid acids that interfere with subsequent molecular techniques [30 (link),36 (link),46 (link)]. A volume of 12 mL of sludge or bacterial suspension was centrifuged for 30 min at 4000× g. The supernatant was discarded, and the pellet was used for DNA extraction using DNeasy^® PowerSoil^® Kit (QIAGEN GmbH., Hilden, Germany) according to the protocol provided by the supplier and a previous study [27 (link)]. The purity, as the concentration of the resulting DNA preparation, was determined spectrophotometrically by the Qubit^® system (Thermo Fisher, Carlsbad, CA, USA). The DNA integrity of molecular weights over 2000 pairs of bases (bp) was evaluated using 2.0% agarose gels (Bioline, London, UK) with Invitrogen SYBR^® Safe DNA gel stain (Thermo Fisher) and TAE buffer solution (40 mol L⁻¹ tris-hydroxymethyl-aminomethane, 20 × 10⁻³ mol L⁻¹ acetic acid, and 1 × 10⁻³ mol L⁻¹ EDTA). Electrophoresis was carried out at 80 mV for 30 min in a Gel XL EnduroTM chamber (Labnet, Edison, NJ, USA) and using 2 µL of DNA per well. Additionally, DNA of E. coli ATCC^® was extracted using an E.Z.N.A^® Bacterial DNA kit (Omega Bio-tek, Norcross, GA, USA), following supplier directions.

For detection of CanineCV in dog samples, a conventional PCR assay was performed using a set of specific primers reported by Kotsias et al.^{25 (link)}. Briefly, the primers Forward For-genomic-(5′-ATGGCTCAAGCTCAGGTTG-3′) and the Rev 533 Reverse primer (5′-CCGCACAGAACCTCCACTTC-3′) were used. To confirm CPV-2-positive samples, PCR amplification of VP2 was conducted using the Forward Ext1F primer (5′-ATGAGTGATGGAGCAGTTCA-3′) and the Ext3R Reverse primer (5′-AGGTGCTAGTTGAGATTTTTCATATAC-3′) described by Ref.^{26 (link)}. In all cases, reactions were performed in a total volume of 25 ul containing 1 unit of Taq polymerase (Go taq flexi-Promega), 1 × Taq buffer, 2 µM MgCl₂, 0.5 µM dNTPs, 20 pmol of each primer and 100 ng of extracted DNA. The PCR reactions were performed on a C1000 Touch BIORAD-DNA thermocycler (Biorad; CA, USA). Molecular grade water was used as a negative control for amplifications. PCR amplification results were visualized using 1.5% horizontal agarose gel electrophoresis stained with the Invitrogen SYBR Safe DNA Gel Stain (Thermo Fisher Scientific). The GeneRuler 100-bp DNA Plus Ladder (Thermo Fisher Scientific) was used as a molecular weight marker. Gels were visualized in the ultraviolet light Gel Doc XR + imaging system (Bio-Rad, Molecular imager, USA) by using ImageLab software.

A PCR method [21 (link)] was performed to determine if isolates belonged to the Legionella genus and L. pneumophila species. These tests were also used to confirm Knoxville-1 33153 isolates. DNA extraction was performed on isolated colonies derived from environmental samples using a ZYMO Research yeast/bacterial DNA extraction kit (Zymo Research Corporation, Irvine, CA, USA). L. pneumophila specific mip gene primers LpneuF (5′-CCGATGCCACATCATTAGC-3′) and LpneuR (5′-CCAATTGAGCGCCACTCATAG-3′), as well as Legionella genus 16S rRNA primers LEG-225 (5′-AAGATTAGCCTGCGTCCGAT-3′) and Leg-858 (5-GTCAACTTATCGCGTTTGCT-3′) were used. The PCR amplification mixture used consisted of 12.5 µL Promega GoTaq Green MasterMix (Promega Biosciences LLC., San Luis Obispo, CA, USA), 10 µL DNA template, and 0.13 µM each primer, with a final reaction volume of 25 µL. Gel electrophoresis was performed in a 1% agarose gel containing 0.05 µL/mL of 10,000X Invitrogen SYBR Safe DNA Gel Stain (Life Technologies Corporation, Carlsbad, CA, USA) to detect PCR products.

DNA extraction was performed on drinking water meter biofilm samples using a ZYMO Research yeast/bacterial DNA extraction kit (Zymo Research Corporation, Irvine, CA, USA). Legionella spp. and L. pneumophila specific primers were used in this study (Table 2). For both primer sets, PCR amplification mixtures consisted of: 12.5 µL Promega GoTaq Green MasterMix (Promega Biosciences LLC., San Luis Obispo, CA, USA), 10 µL DNA template, and 0.13 µM each primer, with a final reaction volume of 25 µL. Gel electrophoresis was performed in a 40 mL 1% agarose gel containing 2 µL of 10,000X Invitrogen SYBR Safe DNA Gel Stain (Life Technologies Corporation, Carlsbad, CA, USA) to detect PCR products. Sequencing was performed on PCR products to confirm the presence of Legionella or L. pneumophila.