A solution of 10X phosphate-buffered saline (PBS) was prepared by dissolving 3.2073 g NaCl, 0.0807 g KCl, 0.5678 g Na2HPO4, and 0.0978 g KH2PO4 in 40 mL in molecular biology grade water (Fisher Scientific) and adjusting the pH to 7.35 with NaOH. Saliva dilution buffer was created by mixing 200 μL 10X PBS, 1400 μL molecular biology grade water (Fisher Scientific), and 400 μL BLUE buffer (MicroGEM International, PLC.). Saliva was diluted in this buffer in a 1:3 ratio. VTM from clinical samples was serially diluted in the resultant mixture prior to PDQeX extraction.
Molecular biology grade water
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Molecular biology grade water is a highly purified water product designed for use in molecular biology applications. It is free of detectable RNase, DNase, and other contaminants that could interfere with sensitive molecular techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bacto eugon agar, by BD (2 mentions)
Difco yeast extract, by BD (2 mentions)
Bbl beef extract, by BD (2 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Pcr buffer, by Qiagen (1 mentions)
Uv transilluminator, by Bio-Rad (1 mentions)
E swabs 480ce, by Copan (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Lithium chloride (licl), by Thermo Fisher Scientific (1 mentions)
Glycine, by Thermo Fisher Scientific (1 mentions)
Bhi broth, by Thermo Fisher Scientific (1 mentions)
Bhi agar, by Thermo Fisher Scientific (1 mentions)
Cytidine 5 monophosphate disodium salt, by Merck Group (1 mentions)
Guanosine 5 monophosphate disodium salt, by Merck Group (1 mentions)
Adenosine 5 monophosphate disodium salt, by Merck Group (1 mentions)
Atto 532, by ATTO-TEC (1 mentions)
Maxima probe rox qpcr master mix 2, by Thermo Fisher Scientific (1 mentions)
Instagene matrix, by Bio-Rad (1 mentions)
18 protocols using molecular biology grade water
1
Saliva Dilution and VTM Extraction
2
Serotype-Specific PCR for Pneumococcus
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These reactions were performed as previously described [37 (link), 38 (link)] except that we utilized DNA purified from NP samples. Briefly, DNA (2 μl) was used as template in 25 μl PCR reactions containing 50 pmol/μl of each of the pair of primers listed below, 2.5 mmol/l of dNTP, 2.5 μl of 10xPCR buffer (Qiagen), 0.1 μl of Qiagen HotStar Taq polymerase, and molecular biology grade water (Thermo). Reactions specifically target single nucleotide polymorphism within the wciP gene and therefore the following primers will amplify a PCR product from: serotype 6A and 6C, primers wciP584gS (5'-ATTTATATATAGAAAAACTGGCTCATGATAG-3' ) and, wciPr (5'-GCGGAGATAATTTAAAATGATGACTAGTTG-3' ), or a PCR product from serotype 6B and 6D with primers wciP584aS (5'-AAGATTATTTATATATAGAAAAACTGTCTCATGATAA-3' ) and wciPr. Cycling parameters were: one cycle at 95°C for 15 min, 35 cycles of 94°C for 30 s, 62°C for 1 min, and 72°C for 1 min; and a final extension of 72°C for 10 min. Products were run on 3% agarose gels, stained with SYBR Safe DNA gel stain (life technologies, Grand Island, NY) and visualized under a UV transilluminator (BioRad, Hercules CA).
3
Isolation and Characterization of Dichelobacter nodosus
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Samples (n = 2,126) were collected from 10 sheep farms situated within Nottingham, Derby and Northampton from animals with varying disease states. Sterile nylon flock swabs (E-swabs 480CE, Copan U.S.A.) were used for the collection of samples from the interdigital space of sheep and stored in liquid Amies solution at 5°C overnight. The swabs were inoculated onto Hoof Agar plates containing 4% w/v Bacto Eugon agar (BD, U.S.A.), 0.5% w/v Difco Yeast Extract (BD, U.S.A.), 1.5% w/v BBL Beef Extract (BD, U.S.A.), 1% Sodium Chloride and 6.6% w/v ovine hoof powder (Parker et al., 2005 (link)) and incubated anaerobically at 37°C. After 7 days plates were visually checked for putative D. nodosus and if present sub-cultured onto reduced agar (2%), hoof agar plates and incubated anaerobically at 37°C. Pure colonies were collected from plates in sterile PBS, washed by centrifugation and resuspended in molecular biology grade water (ThermoFisher, U.K.).
4
RNA Isolation and qRT-PCR Analysis
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RNA was isolated from the tissue, by lysing the small tissue pieces using lysing matrix tubes in (MP Biomedicals) in TRIzol (Ambion Thermo Scientific), followed by phenol-chloroform total RNA isolation (according manufacturer’s instructions). RNA was quantified using the Nanodrop™ 2000c spectrophotometer (Thermo Scientific) and 500 ng of RNA was used for cDNA synthesis with Superscript II (Thermo Scientific). The final cDNA reaction was diluted 1:20 in molecular biology grade water (Thermo Scientific). qRT-PCR was performed on a 7500 Fast Real-Time PCR machine using the SYBRGreen master mix (Applied Biosystems, Belgium). The relative gene expression was calculated by comparing cycle times for target PCR using the following equation: relative gene expression = 2 – (ΔCtsample − ΔCtcontrol).Values are normalized to housekeeping gene RPL-32 expression levels. Primer sequences for collagen type I, collagen type III, biglycan and RPL-32 can be found in Supplemental Table 2 .
5
Isolation and Analysis of Interdigital Bacterial Isolates
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The bacterial isolation, DNA isolation, sequencing and analysis of sequence data in this study are as described previously in [1 ]. A total of 2126 Interdigital swabs (E-swabs 480 CE, Copan U.S.A.) were taken from ewes and lambs. The swabs were stored in liquid Amies solution at 5 °C overnight prior to being inoculation of hoof Agar plates containing 4% w/v Bacto Eugon agar (BD, U.S.A.), 0.5% w/v Difco Yeast Extract (BD, U.S.A.), 1.5% w/v BBL Beef Extract (BD, U.S.A.), 1% sodium chloride and 6.6% w/v ovine hoof powder [16 (link)]. and incubated under anaerobic conditions at 37 °C. Pure colonies were collected from plates in sterile PBS, washed by centrifugation and resuspended in molecular biology grade water (ThermoFisher, UK). Candidate colonies were identified by visual inspection (n = 83 isolates), bacteriodes spp and contaminated samples were identified by sequencing and eliminated from the analysis. Fifty six isolates remained and were available for further analysis.
6
Oral Listeria Infection Model
7
Quantitative SARS-CoV-2 RNA Assessment in Wastewater
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The quantitative assessment of SARS-CoV-2 RNA GC in wastewater samples was performed with the ViroReal® Kit SARS-CoV-2 & SARS (Ingenetix, Vienna, Austria) on a QuantStudio 6 Pro-instrument (Applied Biosystems®, Thermo Fisher Scientific) targeting a highly conserved region of the nucleocapsid protein gene (N gene). A standard curve for absolute quantification was obtained using serial dilutions (101 to 105 copies/reaction) of a synthetic RNA template (positive control - PC) containing the target sequence. The reaction mixture contains 4 µl molecular biology grade water (Thermo Fisher Scientific), 5 µl of Master Mix (Ingenetix), 1 µl of SARS-CoV-2 Multiplex Assay Mix (comprising the primers and the probe; Ingenetix) and 10 µl RNA template. Thermal cycling conditions were as follow: reverse transcription at 50 °C for 15 min, initial denaturation at 95 °C for 20 s, followed by 45 cycles at 95 °C for 5 s and 60 °C for 30 s. All RT-qPCR reactions were performed in duplicates in a total volume of 20 µl/reaction. For each RT-qPCR run, a standard curve containing the five dilution steps in duplicate and two negative controls (molecular biology grade water was added to the reaction mixture) were included.
8
Fabrication of Microfluidic Devices
9
qPCR Detection of Chlamydiaceae 23S rRNA
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The qPCR followed the protocol described by Everett et al. [17 (link)] for the detection of the 23S rRNA gene of the family Chlamydiaceae, with the following modifications: the primers were TQF 5′-GAAAAGAACCCTTGTTAAGGGAG-3′ and TQR 5′-CTTAACTCCCTGGCTCATCATG-3′, and the probe was FAM-CAAAAGGCACGCCGTCAAC-TAMRA. The reaction volume (25 μl) included 12.5 μl of Maxima Probe/ROX qPCR Master Mix – 2× (Thermo Scientific, Waltham, MA, USA), 1.0 μl of each primer at 10 pmol/μl, 0.5 μl of the probe at concentrations 10 pmol/μl, 5 μl of DNA and 5 μl of molecular biology-grade water (Thermo Scientific). The amplification steps were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The poultry samples were analysed in triplicate. A DNA extract from C. muridarum (ATCC VR-123), donated by the Laboratory of Chlamydias and Human Papillomavirus, Virology Institute, School of Medical Sciences, National University of Córdoba, Argentina was used as a positive control and molecular biology-grade water as a negative control. All samples with amplification of the 130-bp segment and with a growth curve exceeding the cycle threshold (automatically calculated) up to cycle 35 were considered positive [17 (link)].
10
Identification of Presumptive L. monocytogenes Isolates
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Total bacterial DNA from the 13 presumptive L. monocytogenes isolates was extracted using the InstaGene Matrix (BioRad Laboratories, Inc., Hercules, CA, USA) according to manufacturer’s instruction. The 16S rDNA gene was amplified by PCR and then sequenced. PCR amplifications were performed using 25 μL of DreamTaq Hot Start PCR Master Mix 2x (Thermo Scientific, Waltham, MA, USA), 0.5 μM fD1 (5′-AGAGAGTTTGATCCTGGCTCAG-3′), 0.5 μM rD2 (5′-TAAGGAGGAGGTGATCCAGCC-3′), 50–100 ng of purified DNA and 19 μL of molecular biology-grade water (Thermo Scientific). PCR mixtures were subjected to several amplification cycles, starting with an initial denaturation cycle (95 °C, 3 min), followed by 35 cycles of denaturation (95 °C, 30 s), hybridization (60 °C, 30 s), elongation (72 °C, 1 min), and ending with a final elongation cycle (72 °C, 5 min) in a thermal cycler (Eppendorf, Hamburg, Germany). The resulting amplicons were then purified using the Nucleospin Gel and PCR Clean-up kit (Macherey-NagelTM, Düren, Germany) and sent to Eurofins Genomics (Ebersberg, Germany) for DNA sequencing. To determine their taxonomic identification, the nucleotide sequences were analyzed using the BLAST nucleotide server of the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/ , accessed on 28 February 2024).
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