E. faecium was grown in BHI medium, bacterial DNA was extracted using alkaline lysis, and the EntF-encoding DNA fragment was amplified in a Mastercycler PCR system (Eppendorf, Belgium). Each reaction was prepared in 10 μL (final reaction volume) containing 2x BioMix (Bioline, Belgium) and 1 μL of DNA 0.5 μM of each primer (EntF*-PCR primers, Additional file
Biomix
Manufactured by Meridian Bioscience
Sourced in United Kingdom, Germany
BioMix is a laboratory equipment product designed for the efficient mixing of biological samples. It provides a reliable and consistent mixing mechanism to ensure thorough homogenization of various liquids and solutions used in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Mycycler thermal cycler, by Bio-Rad (3 mentions)
Nucleospin plant kit, by Macherey-Nagel (2 mentions)
Mastercycler pcr system, by Eppendorf (1 mentions)
Mastercycler pro, by Eppendorf (1 mentions)
Midori green, by Nippon Genetics (1 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
T3000, by Analytik Jena (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Microspin s 300 hr columns, by GE Healthcare (1 mentions)
Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Nucleofast 96 pcr plate, by Macherey-Nagel (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Megalign pro, by DNASTAR (1 mentions)
Mastercycler pro thermal cycler, by Eppendorf (1 mentions)
454 gs flx system titanium chemistry, by Roche (1 mentions)
Nucleofast 96 pcr clean up kit, by Macherey-Nagel (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
21 protocols using biomix
1
Amplification of EntF-encoding DNA in E. faecium
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E. faecium was grown in BHI medium, bacterial DNA was extracted using alkaline lysis, and the EntF-encoding DNA fragment was amplified in a Mastercycler PCR system (Eppendorf, Belgium). Each reaction was prepared in 10 μL (final reaction volume) containing 2x BioMix (Bioline, Belgium) and 1 μL of DNA 0.5 μM of each primer (EntF*-PCR primers, Additional file5 : Fig. S2b). Amplification was performed as follows: 1 cycle of 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. Final elongation was performed at 72 °C for 10 min, after which PCR products were kept at 4 °C. The PCR amplification products were resolved on 1.5% agarose gel.
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E. faecium was grown in BHI medium, bacterial DNA was extracted using alkaline lysis, and the EntF-encoding DNA fragment was amplified in a Mastercycler PCR system (Eppendorf, Belgium). Each reaction was prepared in 10 μL (final reaction volume) containing 2x BioMix (Bioline, Belgium) and 1 μL of DNA 0.5 μM of each primer (EntF*-PCR primers, Additional file
2
HPV L1 Gene Detection Protocol
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The tissue samples were screened using set of consensus primers My9/My11 for amplifying HPV L1 gene fragments, that can detect high-risk strains of HPV [32 (link)]. Amplification was carried out on 20 μl reaction mixtures containing 2X Biomix (Bioline, UK), forward and reverse primers, 2 μl sample and nuclease free water. The PCR reaction mixture was subjected to initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 45s, 47°C for 1min and 72°C for 1 min. The final extension was done at 72°C for 10 min. All possible precautions, including negative controls were taken to minimize cross contamination. The PCR products were observed in 2% agarose gel with ethidium bromide staining.
3
Specificity Testing of PCR Primers
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The specificity of the different PCR primer pairs was tested using a broad range of E. coli strains (mainly, but not exclusively, APEC), including 5 aromatic amino acid auxotrophic strains (E. coli strains harboring the WT aroA gene, but that are unable to synthesize aromatic amino acids), as well as other non-Escherichia strains (S1 Table ). PCR was performed in a 20 μl total reaction mixture using 2 μl template DNA, 0.5 μM forward and 1 μM reverse primer in case of aroA primers; 0.5 μM in case of any other reverse primer and 2x biomix (Bioline, London, UK). Thermocycling was carried out in an Eppendorf Mastercycler Pro (Eppendorf, Hamburg, Germany) with the following parameters: 1 cycle at 94°C for 10 min followed by 30 cycles at 94°C for 45 s, 60°C for 30 s and 72°C for 45 s followed by 1 cycle at 72°C for 5 min. Agarose gel electrophoresis of the PCR products was performed on a 1.5% (w/v) agarose gel stained with MidoriGreen (Nippon Genetics, Tokyo, Japan) and visualized with UV-light. A 100-bp DNA ladder (GeneRulerTM, Thermo Scientific, Waltham, MA, USA) was used as a molecular weight marker.
4
Extracting and Sequencing rbcL DNA Barcode
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Total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, United Kingdom) in accordance with the manufacturer’s instructions. The rbcL sequences were amplified with rbcLa-F: 5′-ATGTCACCACAAACAGAGACTAAAGC-3′ and rbcLa-R: 5′-GTAAAATCAAGTCCACCRCG-3′; DNA barcoding primers (Hollingsworth, 2011 (link)). PCR used a total volume of 20 μL with 10 μL BioMix (BioLine, United Kingdom), 1 μL of each primer (10 μM) and 1 μL of the DNA template (50 ng/µL) and 7 μL distilled water. PCR involved 1 cycle (94°C for 3 min), 35 cycles (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min), and 1 cycle (72°C for 7 min). The resulting 600 bp band was sequenced using a 3730xl DNA Analyzer (Applied Biosystems, United Kingdom), with the same primers used for PCR. The derived sequences were assessed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi ). The rbcL sequence was submitted to GenBank (accession number: MN431197).
5
ChIP-PCR Analysis of GlnR Binding
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To identify enrichment in GlnR‐immunoprecipitated DNA, a rate‐limiting PCR was performed. DNA was immunoprecipitated and purified as described under ChIP. DNA sequences were amplified using primers listed in Table S1 . Reaction mixtures consisted of GlnR‐immunoprecipitated DNA (0.3 ng), 1 × BioMix (Bioline), 1 μM of each primer and 5% (v/v) dimethyl sulfoxide (Sigma). PCR was carried out in a thermocycler T3000 (Biometra); 95°C for 5 min, 23 cycles of 95°C 30 s, 55°C 30 s, 72°C 1 min, with final extension 72°C for 8 min. DNA was visualised on a 2% agarose gel.
6
Blastocystis Subtyping by Barcoding
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Blastocystis was initially detected and subtyped by the Blastocystis barcoding method previously described, using primers RD5 (5′-ATC TGG TTG ATC CTG CCA GT-3′) and BhRDr (5′-GAG CTT TTT AAC TGC AAC AAC G-3′) [29 (link)]. One μl DNA was used in a reaction mixture of 20 μl Biomix (Bioline, London, UK), each primer at 250 nM, and NF-water to a total volume of 40 μl. Amplification included an initial step at 94 °C for 5 min followed by 30 cycles of denaturation, annealing and extension at 94 °C, 59 and 72 °C (1 min each) and was completed by a final extension at 72 °C for 2 min. All negative samples were run an additional time with the DNA diluted 1/10 before considered negative. Positive PCR products were purified using illustra MicroSpin S-300 HR columns (GE Healthcare, Little Chalfont, UK). Purified PCR products were sequenced using the primer BhRDr, ABI BigDye terminator kit version 1.1 and an ABI 3130xl Genetic Analyzer (Applied Biosystems). Sequencing chromatograms were analysed in the software Chromas Lite version 2.1.1 (Technelysium, Brisbane, Australia). Subtypes were identified by BLAST searches at the National Center for Biotechnology Information (NCBI) determining the exact match or closest similarity to Blastocystis sequences previously deposited in GenBank. The nomenclature for subtypes presented by Stensvold et al. [12 ] was used.
7
Amplifying Plant DNA Barcodes
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Reference barcode PCRs were conducted in 25 μl mixtures comprising: 1x Biomix (Bioline); 200 pM of primers (ndhJ1+ndhJ4 [24 (link)]; rbcLa_f + rbcLa_rev, [25 (link),26 (link)] and 25 ng of DNA. PCR conditions were: 94°C for 30 s, 40 cycles of 94°C 30 s, 54°C 60 s, 72°C 60 s and a final extension of 72°C 5 min. Products were purified using Nucleofast 96 PCR plates (Macherey-Nagel) and Sanger-sequenced. The forward and reverse reads were aligned, edited and trimmed in Geneious (V8.1, Biomatters). Further reference barcodes were recovered from the NCBI database for food plants of captive animals (S1 Text ).
8
Quantification of SMPD1 Splice Isoforms
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Detection and quantification of SMPD1 splice-isoforms were conducted with 5’-6-carboxyfluorescein (FAM)-labeled primers and subsequent capillary electrophoresis with laser-induced fluorescence measurements as previously described [16 (link)]. Amplification of SMPD1 transcripts was carried out with BioMix (Bioline) and the primer sets 5’-FAM-TCCTGGGGCCAGTGCCAG-3’ and 5’-CAGCTCTTCAGACAGTGCC-3’ spanning the exons 2–4 as well as 5’-CAGGATGTAGGTCTCATGGTC-3’ and 5’- FAM-GCTGGAGCTGGAATTATTACC-3’ for exons 4–6, respectively [12 (link)].
9
Mealybug COI Gene Amplification Protocol
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Mealybug genomic DNA extraction was performed using the DNeasy Blood and Tissue kits (Qiagen) according to the manufacturer’s protocol. For each of the extracted genomic DNA sample, a 25 μl PCR reaction mixture was prepared. In the negative control sample, ddH2O was added instead of the DNA template. Primers were designed from the available pseudococcid sequences to amplify a 379-bp partial region of the COI gene (Wetten et al., 2016 (link)). Each PCR reaction mixture contained 2 μl genomic DNA, 2.5 μl COI forward primer (2 μM) (Table 2 ), 8 μl ddH2O, and 12.5 μl BioMix (Bioline). All PCR reactions were performed in a G-STORM GS2 thermal cycler (Gene Technologies Ltd.). The first step (5 cycles) included initial DNA denaturation for Taq activation at 94°C for 1 min, annealing at 46°C for 1 min 30 s, and elongation at 72°C for 1 min 30 s. The second step (35 cycles) included denaturation at 94°C for 1 min, annealing at 50°C for 1 min 30 s, and elongation at 72°C for 1 min, with a final elongation at 72°C for 5 min and storage at 10°C for 30 s. The PCR products were assessed by 1% agarose gel electrophoresis (as described above). The PCR products were Sanger sequenced on both strands by Source BioScience Ltd.. Consensus sequences were produced, and alignments were manually edited with Geneious 9.0.
10
Genome-Wide DNA Damage Assessment via RAPD
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Random Amplification of Polymorphic DNA (RAPD) was used to analyze the DNA damage sustained by the whole genome. It was performed using BioMix™ (BioLine GmbH, Luckenwalde, Germany), by adding 1 μL (5 pM) primer to 1 ng template DNA and making it up to a final volume of 25 μL using distilled water. The primer used for RAPD was GGAGGAGGAGGAGGAGGAGGA (GGA7) [37 (link)]. Amplification was carried out using an initial denaturation step for 2 min at 94 °C, followed by denaturation for 20 s at 94 °C, annealing for 60 s at 49 °C, and extension for 20 s at 72 °C. The last three steps were each repeated 40 times, with a last extension for 6 min at 72 °C. Molecular approaches based on PCR-stop (see above) and genomic PCR fingerprinting assays are powerful tools for evaluating DNA damage; the protocols used here were validated for microorganisms [3 (link),20 (link),38 (link),39 (link)] and optimized for fungi [22 (link)]. These approaches have been already applied to assess DNA damage in a number of studies carried out on both ground-based and space experiments on dried colonies of Cryomyces [20 (link),21 (link),24 ].
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