The presence of genes commonly associated with erythromycin resistance (ermA and ermB) and tetracycline resistance (tetK/L, tetM and tetO) was assessed for the streptococci strains using PCR with the primers and conditions presented in Table 3 . Universal primers that detect tetracycline resistance genes encoding ribosome protection proteins were used first. Then, in the case of the isolates that tested positive in this reaction, the specific primers for tetM and tetO were used. All PCR mixtures contained 1 µL of each primer (10 pmol/µL), 12.5 µL of DreamTaq Green PCR Master Mix (2×) (Thermo Fisher Scientific, Waltham, MA, USA), 40 ng of DNA template and nuclease-free water up to 25 µL. Reaction products were recognized by electrophoresis in 1% (w/v) agarose gel in Tris-Acetate-EDTA (TAE) buffer with Midori Green DNA Stain (Nippon Genetics, Düren, Germany), visualized and analyzed using a Gel DocTM EZ Imaging System with Image Lab Software (version 5.2.1) (Bio-Rad, Hercules, CA, USA).
Midori green dna stain
Manufactured by Nippon Genetics
Sourced in Germany, Japan
Midori Green DNA Stain is a fluorescent dye used for the detection and visualization of DNA in gel electrophoresis and other nucleic acid analysis applications. It binds to double-stranded DNA, emitting a green fluorescent signal when exposed to ultraviolet (UV) or blue light.
Automatically generated - may contain errors
Lab products found in correlation
Dreamtaq green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Gel doctm ez imaging system, by Bio-Rad (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Dreamtaq green pcr master mix 2, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Gel out, by A&A Biotechnology (1 mentions)
Pcr thermal cycler dice, by Takara Bio (1 mentions)
Quick taq hs dyemix, by Toyobo (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Generuler 100 bp plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Gotaq flexi dna polymerase, by Promega (1 mentions)
High pure viral nucleic acid large volume kit, by Roche (1 mentions)
Rnase 1, by Thermo Fisher Scientific (1 mentions)
Miseq reagent kit v2 500 cycle, by Illumina (1 mentions)
Miseq next generation sequencing platform, by Illumina (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Takara taq hs, by Takara Bio (1 mentions)
Mastercycler nexus, by Eppendorf (1 mentions)
19 protocols using midori green dna stain
1
Genetic Detection of Antibiotic Resistance
2
Nested PCR for Screening of aHEV Samples
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For the screening of aHEV-positive samples, the nested polymerase chain reaction (PCR) assay was performed with two degenerate primers sets that targeted the partial helicase gene region in ORF1, as described previously [13 (link), 17 (link)]. A previously sequenced aHEV fragment was used as a positive control (GenBank accession number MH636899.1). The size of the obtained PCR product was 386 bp.
The products resulting from second-round PCR were examined on a 2% agarose gel stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Düeren, Germany). The amplified products of ORF1 were excised, purified using Gel-Out (A&A Biotechnology, Gdynia, Poland), and directly sequenced in both directions with Sanger’s method (Eurofins Genomics Sequencing GmbH, Cologne, Germany) with the use of PCR primers.
The products resulting from second-round PCR were examined on a 2% agarose gel stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Düeren, Germany). The amplified products of ORF1 were excised, purified using Gel-Out (A&A Biotechnology, Gdynia, Poland), and directly sequenced in both directions with Sanger’s method (Eurofins Genomics Sequencing GmbH, Cologne, Germany) with the use of PCR primers.
3
PCR Detection of Enteric Viruses
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Two microliters of sample cDNA was mixed with 25 μl of Quick Taq HS DyeMix (Toyobo, Japan), 1 μl of 20 μM primer mix (P290d: 5′-GATTACTCCASSTGGGAYTCMAC-3′, P289d: 5′-TGACGATTTCATCATCMCCRTA-3′) (Pinto et al., 2012 (link)), and 22 μl of distilled water. Using a PCR Thermal Cycler Dice (TaKaRa, Japan), the DNA was amplified at 94 °C for 5 min, followed by 36 cycles of denaturation at 94 °C for 30 s, primer annealing at 45 °C for 30 s, and synthesis at 72 °C for 60 s, with a final extension at 72 °C for 10 min. PCR products (5 μl) were electrophoresed with a DNA marker on a 1.5% agarose gel. The agarose gel was incubated with Midori Green DNA Stain (Nippon Genetics, Japan), and bands of 319 bp (in case of NoV) and 331 bp (in case of Vesuvius and Sapovirus) were visualized using a UV Transilluminator. The methods used to detect viral genes other than FNoV were described previously (Chung et al., 2013 (link), Elschner et al., 2002 (link), Martella et al., 2012 (link), Pinto et al., 2012 (link), Schunck et al., 1995 (link), Takano et al., 2013 (link)).
4
Rapid Enterococcus Species Identification
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A single PCR method was used to identify genus Enterococcus, whereas the species level of E. faecalis, E. faecium, or E. gallinarum was determined in multiplex PCR. The primer sequences, specific for simultaneous amplification of the ddl gene (E. faecalis and E. faecium) and sodA gene (E. gallinarum), are listed in Table 2 . The protocols of the single and multiplex PCR were described by Ke et al.27 (link) and Yean et al.,29 (link) respectively. Strains used as positive controls were as follows: E. faecalis ATCC 51299, E. faecium ATCC 700221, and E. gallinarum ATCC 700425. Products obtained by amplification were divided by electrophoresis in 2% agarose gels. DNA bands were stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Dueren, Germany) and visualized with an ultraviolet transilluminator.
5
Genomic DNA Extraction and PCR Detection
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DNA Extraction
Total genomic DNA was obtained using the Syngen DNA Mini Kit (Syngen Biotech Sp. z o.o., Wrocław, Poland). The amount and quality of DNA was determined using the Thermo Scientific NanoDropTM 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Polymerase Chain Reaction (PCR)
The B. cereus isolates were analyzed for the presence of the emetic toxin gene using the primers CesF1 and CesR2 [23 (link)]. The reaction mixture contained 10 μM of each primer, 12.5 μL of DreamTaq PCR Master Mix (2×) (Fermentas, Thermo-Fisher Scientific Inc., Waltham, MA, USA), 100 ng DNA and water up to 25 μL. The following amplification procedure was used: Initial denaturation at 94 °C for 4 min., 40 cycles of 94 °C for 30 s, 54 °C for 45 s, 72 °C for 1 min and the final extension step at 72 °C for 7 min. The PCR product in a total volume of 15 μL was separated in 1.0% agarose gel stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Dueren, Germany). GeneRuler 100 bp Plus DNA Ladder was used for estimating the molecular size weight of the obtained band (Fermentas, Thermo-Fisher Scientific Inc.). The DNA from the reference strains was used as positive control—B. cereus NCTC 11143 and negative control—B. cereus ATCC 14579.
Total genomic DNA was obtained using the Syngen DNA Mini Kit (Syngen Biotech Sp. z o.o., Wrocław, Poland). The amount and quality of DNA was determined using the Thermo Scientific NanoDropTM 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Polymerase Chain Reaction (PCR)
The B. cereus isolates were analyzed for the presence of the emetic toxin gene using the primers CesF1 and CesR2 [23 (link)]. The reaction mixture contained 10 μM of each primer, 12.5 μL of DreamTaq PCR Master Mix (2×) (Fermentas, Thermo-Fisher Scientific Inc., Waltham, MA, USA), 100 ng DNA and water up to 25 μL. The following amplification procedure was used: Initial denaturation at 94 °C for 4 min., 40 cycles of 94 °C for 30 s, 54 °C for 45 s, 72 °C for 1 min and the final extension step at 72 °C for 7 min. The PCR product in a total volume of 15 μL was separated in 1.0% agarose gel stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Dueren, Germany). GeneRuler 100 bp Plus DNA Ladder was used for estimating the molecular size weight of the obtained band (Fermentas, Thermo-Fisher Scientific Inc.). The DNA from the reference strains was used as positive control—B. cereus NCTC 11143 and negative control—B. cereus ATCC 14579.
6
Characterization of Novel CYP2A6 Variant
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A novel SV was identified, designated CYP2A6*53 by PharmVar (31) . A CYP2A6*4/*53 individual was selected for characterization of CYP2A6*53 using nested PCR and Sanger sequencing. The first PCR reaction amplified CYP2A6 from exon 3 to the 3' flanking region (NC_000019.9:g.41347784_41354528) using CYP2A6-specific primers (Supplementary Table 2). Two subsequent nested PCR reactions were specific for CYP2A6*53: first, using a CYP2A6-specific forward primer in exon 4 and CYP2A7-specific reverse primer in Intron 7 (generating the intron 4 product); second, using a CYP2A7-specific forward primer in Intron 7 and CYP2A6-specific reverse primer in the 3' flanking region (generating the 3' product)(Figure 1). Amplification of both products was observed in the CYP2A6*4/*53 target, while no amplification of CYP2A6*53 was observed in a CYP2A6*4/*4 control.
The intron 4 and 3' products were extracted from 0.8% agarose gels with Midori Green DNA stain (Nippon Genetics Co., Ltd., Tokyo, Japan) using a QIAquick gel extraction kit (QIAGEN, Hilden, Germany).
The intron 4 product was sequenced for intron 4 and exons 5-7, while the 3' product was sequenced for exons 8-9, and the 3'-UTR and flanking region. Sequencing primers were designed to be CYP2A6-, CYP2A7or CYP2A6/7-specific as described in Supplementary Table 2.
The intron 4 and 3' products were extracted from 0.8% agarose gels with Midori Green DNA stain (Nippon Genetics Co., Ltd., Tokyo, Japan) using a QIAquick gel extraction kit (QIAGEN, Hilden, Germany).
The intron 4 product was sequenced for intron 4 and exons 5-7, while the 3' product was sequenced for exons 8-9, and the 3'-UTR and flanking region. Sequencing primers were designed to be CYP2A6-, CYP2A7or CYP2A6/7-specific as described in Supplementary Table 2.
7
Detection of DNMT3A R882H Mutation
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DNMT3A gene mutation (R882H; GCCGC to GCCAC) was genotyped by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Genomic DNA was amplified in a 20 μl volume containing 1 U of GoTaqFlexi DNA polymerase (Promega, Poland), 1 μl of template DNA, 4 μl of 5X PCR buffer, 2.0 mM MgCl2, 0.3 mM dNTPs (mixture of dATP, dTTP, dCTP and dGTP), and 0.6 pmol of each primer (forward: 5′-GTGATCTGAGTGCCGGGTTG-3′ and reverse: 5′-TCTCTCCATCCTCATGTTCTTG-3′). The PCR cycling conditions were: 10 min of initial denaturation at 95°C, followed by 35 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 59°C and extension for 1 min at 72°C, followed by 7 min of final extension at 72°C. A 7 μl aliquot of PCR product was then digested with 3 U of restrictive enzyme SatI (Fnu4HI) and 2 μl of 1X BufferG (Thermo Scientific) in a final volume of 20 μl. After incubation at 37°C for 16 h restriction fragments were separated in a 2% agarose gel stained with Midori Green DNA Stain (Nippon Genetics) and visualized under UV light. Positive samples showed 3 bands (289 bp, 190 bp, 114 bp) because of the loss of a restriction site of SatI caused by the mutation.
8
Phage DNA Extraction and Sequencing Protocol
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High-titer phage suspensions (1012–1014 PFU/ml) were used for DNA extraction. Prior to the extraction, bacterial lysates containing phages were treated with DNase I (80 U/ml; Thermo Scientific, USA) and RNase I (80 μg/ml; Thermo Scientific, USA) at 37 °C for 3 h to remove non-phage nucleic acids. Phage DNA was then extracted using a High Pure Viral Nucleic Acid Large Volume Kit (Roche, Mannheim, Germany) with initial phage capsid disruption by treatment with proteinase K and 0.5% sodium dodecyl sulfate (Sigma-Aldrich) for 2 h at 56 °C. The integrity of the extracted DNA was determined by electrophoresis in 0.7% agarose stained with Midori Green DNA Stain (Nippon Genetics Europe, Germany). The concentration of DNA was determined with a Biowave II UV/Vis spectrophotometer (Biochrom WPA, UK) and purity was determined in terms of the ratio 260/280 nm. Phage genomes were sequenced with the Illumina MiSeq next-generation sequencing platform (Genomed SA. Poland) using MiSeq Reagent Kit v2 500-cycles (Illumina, USA). Sequencing quality was assessed on the basis of average base quality, GC content and adapter contamination [31 ]. All sequenced phages were assembled into one unique contig and sequence assembly was conducted with the Shovill pipeline and assembly improvement pipeline [32 ]. Genome assemblies were annotated with Prokka [33 ].
9
RT-PCR Amplification and Gel Detection
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RT-PCR was performed in a 10-μl volume containing 45 ng cDNA, 1 µl of 10 × PCR buffer, 0.2 mM of dNTP, 0.25 units TaKaRa Taq
HS (Takara Bio), and 250 nM of each specific primer (Supplemental Table 2 : on-line only). All primer pairs were designed to
span introns to prevent amplification of products from genomic DNA. PCR products were electrophoresed using 1.5% agarose gel
and were detected by Midori Green DNA Stain (Nippon Genetics, Tokyo, Japan).
HS (Takara Bio), and 250 nM of each specific primer (
span introns to prevent amplification of products from genomic DNA. PCR products were electrophoresed using 1.5% agarose gel
and were detected by Midori Green DNA Stain (Nippon Genetics, Tokyo, Japan).
10
PCR Amplification of A. lumbricoides β-tubulin
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PCR amplification was performed with the primers Al167F 5′-GCG GTC ATA GTT TTC AGG GTT T-3′/Al198R 5′-CTC CGT ATG TGG GAT TTG TAA GC-3′ (608 bp) designed on the basis of the A. lumbricoides β-tubulin nucleotide sequence [6 (link)]. The 25 µL of PCR mixture contained 12.5 µL DreamTaq Green PCR Master MIX (Thermo Scientific, Waltham, MA, USA), 1 µL of each primer at the concentration of 10 µM, 5.5 µL nuclease-free water and 5 µL of the DNA extracted from each sample. The PCR thermal cycle conditions were as follows: an initial denaturation step took place at 95 °C for 2 min; it was followed by 40 cycles of amplification for 30 s at 94 °C, 45 s at 60 °C and 1 min at 72 °C. Final elongation was performed at 72 °C for 8 min. All reactions were carried out in a Mastercycler Nexus (Eppendorf, Hamburg, Germany). Amplification products were visualized by electrophoresis on 1.5% agarose gel stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Düren, Germany). In each PCR run, nuclease-free water as a negative control and positive control samples were used. The positive control for the A. suum β-tubulin gene included genomic DNA from a previously positive sample.
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