The enterobacteriophage MS2 (DSM No 13767; strain PC-V3463; Genbank NC_001417 ) and Escherichia coli host cells (DSMZ 5695) were purchased (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig). A stock of MS2 was prepared of 4.8 × 1010 PFU per mL. The MS2 genome was isolated using the NucliSENS miniMAG kit (bioMérieux; Benelux, Zaltbommel, Netherlands) and after treatment with DNase converted into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). The cDNA was used undiluted (approx. 2 × 106 PFU) or diluted in 10-fold steps down to 10−10 and analyzed in 20 μL reactions using iQ™ Supermix (Biorad) or SsoFast™ EvaGreen® Supermix (Biorad) and primers at various concentrations in checkerboard designs. The qPCR was run in a BioRad CFX96™touch Real-time PCR detection system: 10 min 95 °C followed by 50 cycles of 15 s 95 °C and 1 min 60 °C, and a melting curve analysis to verify the amplification of the correct product. Each PCR variable was tested in one or more runs to reach at least triplicate measurements
Nuclisens minimag kit
Manufactured by bioMérieux
Sourced in Netherlands, France
The NucliSENS miniMAG kit is a compact, automated system designed for the extraction and purification of nucleic acids (DNA and RNA) from a variety of sample types. The system utilizes magnetic silica technology to efficiently capture and isolate the target nucleic acids, which can then be used in downstream analytical applications.
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Lab products found in correlation
Qiazol lysis reagent, by Qiagen (2 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Iq supermix, by Bio-Rad (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
Peg 8000, by Merck Group (1 mentions)
Rnasin plus rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
7 protocols using nuclisens minimag kit
1
Quantitative Detection of MS2 Bacteriophage
2
Extraction and Purification of MRV RNA
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For MRV detection, ca 50 g of tissue were taken from each liver. They were finely cut and 450 mg of each sample, were weighed and divided into three sterile 2 mL tubes each containing 150 mg of liver, 1.5 mL of QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and 2 glass beads (5 mm diameter). Then, 10 μL of Mengovirus (recombinant Mengovirus—vMC0; 104 viral particles/μL) were spiked into every sample as process control, as suggested by the UNI CEN ISO/TS 15216 2:2013). The samples were homogenized with the TissueLyser (Qiagen, Hilden, Germany) at 30 Hz for 10 min. After incubation at room temperature for 5 min, 0.2 mL of Chloroform (1 M) were added to each tube. Samples were left at room temperature for 5 min and after centrifugation at 7000× g for 3 min at 4 °C, the supernatant was ready for RNA purification. Viral RNA was purified using the NucliSENS® MiniMag kit (bioMérieux SA, Marcy-l’Etoile, France) according to the manufacturer’s instructions. The eluted RNA was stored at −80 °C until use.
3
Disrupting Toxocara canis Eggs
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Six methods for disruption of T. canis eggs (1, 10, 102 and 103 egg suspensions) were compared: (i) enzymatic lysis with proteinase K (PK) [incubation of egg solution with 0.2 unit of proteinase K in 40 µl solution containing 10% (w/v) of SDS at 56 °C under agitation at 800 rpm for 2 h using a thermomixer (Eppendorf, Hamburg, Germany)]; (ii) thermal disruption (TD) (5 freeze–thaw cycles: 3 min of freezing in liquid nitrogen, followed by 3 min of thawing in boiling water under agitation at 800 rpm in a thermomixer); (iii) mechanical disruption of eggs using FastPrep® tubes containing the lysing matrix A beads (FPA) (MP Biomedicals, Santa Ana, CA, USA) under shaking at 6 m/s for 40 s in a FastPrep-24 homogenizer (three cycles); (iv) the same protocol as the previous but using lysing matrix D beads (FPD) instead; (v) TD followed by FPD (TD-FPD); and (vi) TD-FPD followed by PK (TD–FPD-PK). Following the disruption step, DNA was extracted using the NucliSENS® MiniMag® Kit (bioMérieux, Boxtel, Netherlands) according to the manufacturer’s protocol. DNA solutions were stored at −20 °C until use.
4
Viral RNA Extraction and Purification
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Viral RNA was extracted and purified using the NucliSENS® MiniMag kit (bioMérieux SA, Marcy-l’Etoile, France) according to the manufacturer’s instructions, as suggested by ISO/TS 15216-2:2013 [53 ]. A negative extraction control was processed with every run of extraction. The eluted RNA was stored at −80 °C until use.
5
Norovirus Extraction and RNA Isolation
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The noroviruses were extracted from the spiked lettuce subsamples using the ISO 15216-1:2017 method [5 ]. The capsid integrity experiments were performed on the 500 µL PBS solubilized polyethylene glycol (PEG 8000) (Sigma-Aldrich, Oakville, ON, Canada) pellet. After the capsid integrity treatments, the NucliSens miniMAG kit (Biomérieux, Montréal, QC, Canada) was used to extract the RNA following the manufacturer’s recommendations. RNA was eluted in 100 µL RNase-free water and 1 μL of RNasinTM Plus RNase Inhibitor (ThermoFisher, Asheville, NC, USA) was added to the eluate before its storage at −80 °C.
6
Liver Sample RNA Extraction
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For each liver sample 450 mg were homogenized with QIAzol Lysis Reagent (Qiagen, Hilden, Germany) according to producer instructions and spiked with 10 μL of a titrated suspension of Mengovirus process control (1.6 × 105 TCID50 per ml; strain MC0). RNA has then been purified with the NucliSENS® MiniMag Kit (bioMérieux, Marcy-l’Étoile, Francia). The eluted RNA was conserved at -80 °C until use.
7
Viral RNA Extraction for NoV and HAV
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Viral RNA was extracted following the ISO/TS 15216-2:2013 method for NoV and HAV detection in foodstuffs. In brief, 25g of each sample were cut into small pieces and homogenized with TGBE buffer pH 9.5 (100 mM Tris-HCl, 50 mM glycine, 1% beef extract) and 10 μl of process control virus material (Mengovirus). The eluate was concentrated with 5X PEG/NaCl solution (50% (w/v) PEG 8000, 1.5 M NaCl) and the viral nucleic acids were extracted and purified using commercial kits (NucliSENS miniMAG kit, bioMérieux) according to the manufacturer's instructions.
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