Forty bivalve juveniles of Pinna spp., captured in the wild and distinguished from the other mollusks by morphological characteristics were hosted each one during 24 hours in separate aquaria. Water samples (200 ml each) from each aquarium were filtered through a 0.45 µm screen. The dry filters were conserved in Eppendorfs and stored at -80°C. DNA extractions were carried out by DNeasy PowerWater kit (Qiagen), according to the manufacturer's instructions, using a half filter of each sampling (the other half was stored). DNA quality and quantity were tested with a Nanodrop 2000 instrument (ThermoScientific) and used for multiplex-PCR. For samples used as controls, mantle tissues from P. rudis and P. nobilis adult dead individuals were collected. Total DNA was extracted from mantle using the NucleoSpin® Tissue DNA Isolation Kit (Macherey-Nagel), according to the manufacturer's instructions.
Nucleospin tissue dna isolation kit
Manufactured by Macherey-Nagel
Sourced in Germany
NucleoSpin® Tissue DNA isolation kit is a lab equipment product designed for the isolation of genomic DNA from various tissue samples. The kit utilizes a silica-based membrane technology to efficiently capture and purify DNA, making it suitable for a range of downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 instrument, by Thermo Fisher Scientific (1 mentions)
Dneasy powerwater kit, by Qiagen (1 mentions)
Telotaggg telomere length assay kit, by Roche (1 mentions)
Mx3000p thermocycler, by Agilent Technologies (1 mentions)
Qiasymphony, by Qiagen (1 mentions)
Qiasymphony dsp virus pathogen kit, by Qiagen (1 mentions)
Mastercycler, by Eppendorf (1 mentions)
Gel documentation system, by Bio-Rad (1 mentions)
7 protocols using nucleospin tissue dna isolation kit
1
Bivalve Juvenile Diversity Profiling
2
Telomere Length Measurement Protocol
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Total genomic DNA (gDNA) was isolated from cells with the Nucleospin tissue DNA isolation kit (Macherey Nagel, Germany). The relevant telomeres’ length in different cells was measured with the TeloTAGGG telomere length assay kit (Roche, USA) according to manufacturer instructions; specifically, 2 μg gDNA/sample was initially digested with the Hingl/RSA1 system, then electrophoresed in 1% agarose/TAE gel, and transferred for southern blot on positively charged nylon membrane at 20 X potassium citrate buffer. The gDNA hybrid was tagged with a non-isotopic marker labeled with digoxygen (DIG) incubated with an antibody against DIG covalently linked to alkaline phosphatase and visualized with an optical analysis system chemiluminescence (ECL) (MicroChemi, Israel). The length of the telomeres was evaluated with final restriction fragment analysis (TRFs, terminal restriction fragments) calculated as follows: TRFs = Σ (ODi) / Σ (ODi / Li), where ODi is the intensity of the chemiluminescent signal and Li was the length of the TRF at position i. TRF analysis was done with a special software TeloTool (Göhring et al. 2014 ).
3
Duplex qPCR Detection of Mycobacterial Pathogens
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Presence of MTC and MAC was tested by an in-house duplex qPCR [6 (link)]. Briefly, fat and connective tissue were removed from samples and up to 2 g of tissue were homogenised in a stomacher with 10 ml of sterile distilled water for 2 min. The obtained solution was centrifuged for 10 min at 1400 g resulting in a pellet for each sample. Genomic DNA was extracted from 25 mg of tissue homogenate using NucleoSpin® Tissue DNA isolation kit (Macherey-Nagel GmbH, Düren, Germany) according to the manufacturer’s instructions.
qPCR reactions were run in duplicate in a Agilent Technologies Mx3000P thermocycler under the following conditions: initial denaturation at 95 °C for 10 min, 40 cycles of amplification consisting of denaturation at 95 °C for 30 s, primer annealing at 65 °C for 30 s, and extension at 72 °C for 30 s. DNA from M. bovis and M. avium isolates and non-template controls were included in each assay and used as positive and negative controls, respectively.
qPCR reactions were run in duplicate in a Agilent Technologies Mx3000P thermocycler under the following conditions: initial denaturation at 95 °C for 10 min, 40 cycles of amplification consisting of denaturation at 95 °C for 30 s, primer annealing at 65 °C for 30 s, and extension at 72 °C for 30 s. DNA from M. bovis and M. avium isolates and non-template controls were included in each assay and used as positive and negative controls, respectively.
4
Fecal DNA Extraction Protocol
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Fresh stool specimens were collected from all 50 participants on site during a study visit at Kavar (University cohort center) and Motahari outpatient clinic. Each fecal sample was immediately transferred on ice and were stored at − 80 °C. Then, total DNA was extracted from the samples using a NucleoSpin® Tissue DNA isolation kit (Genomic DNA from tissue, Macherey-Nagel, Germany), following the manufacture’s modified protocol for microorganism DNA from the stool.
5
Quantifying Cell-free and Cellular DNA
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Cellular DNA, collected an hour after reperfusion and at day 14, was isolated from 10 mg tissue or cell pellets using a NucleoSpin Tissue DNA isolation kit (Macherey-Nagel) according to the manufacturer's protocol. Cellfree DNA from 1 mL of plasma was isolated using a QIAsymphony and a QIAsymphony DSP Virus/Pathogen Kit (Qiagen) according to the manufacturer's protocol. Y-chromosome DNA was detected by quantitative polymerase chain reaction (qPCR) using primers for the malespecific repeat located on the porcine Y-chromosome. 29 The porcine S100C gene was used as pig DNA control. Primer sequences are listed in Table S1 (SDC, http://links. lww.com/TP/C7). The qPCR mix consisted of 0.5 µL (cellular) or 5 µL DNA (cell-free), 10 pmol of each primer, and 1× KiCStart SybrGreen qPCR ReadyMix (Sigma-Aldrich Life Science) in an end volume of 25 μL, and run in duplicate on an Applied Biosystems 7300 Real-Time PCR machine (ThermoFisher).
6
Leptospira Detection in Water Samples
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The pH and temperature of the samples were noted at the time of collection. Other physiochemical parameters viz. turbidity, salinity, conductivity and dissolved oxygen were recorded by using Multiparameter water analyser (Thermo Fisher Scientific, Singapore). All the water samples collected were subjected to PCR as per the procedure explained by Tansuphasiri et al. (2006) and Vishak (2015) . The Leptospira DNA isolation was carried out using Nucleospin Tissue DNA isolation kit (Macherey-Nagel, Germany). Four oligonucleotide primers targeting the 16Sr RNA, lipl 32, lipl 21 and lipl 41 were used in the study for detection of Leptospira organism. The PCR amplification was carried out in an automated thermal cycler (Eppendorf Master Cycler, Germany) . The detection of PCR product was carried out by electrophoresis in 1.5 per cent agarose gel in Tris Boric acid EDTA (TBE) electrophoresis buffer (1X). The gel was visualized under UV transilluminator and the images were documented on gel documentation system (Bio-Rad Laboratories, USA). The data obtained were subjected to statistical analysis using the SPSS software version 21.
7
Quantitative Detection of Porcine Y-Chromosome
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DNA was isolated from 10 mg of kidney tissue or from cell pellets from the adherent fraction using a NucleoSpin Tissue DNA isolation kit ( Macherey-Nagel, Du ¨ren, Germany) according to manufacturer's protocol. DNA was eluted in 100 mL water, and Y-chromosome was detected by quantitative polymerase chain reaction using primers directed to the male specific repeat ( MSR) located on the porcine Y-chromosome as previously done by Gruessner et al. [31] .
Primers directed to porcine S100C gene were used as a pig DNA control. Primer sequences were as follows: MSR forward 5¢-CCA TCG GCC ATT GTT TTC CTG TTC A-3¢, MSR reverse 5¢-CCT CTG TGC CCA CCT GCT CTC TAC A-3¢, S100C forward 5¢-ATG CTG GAA GGG ACG GTA ACA ACA-3¢, and S100C reverse 5¢-GCT CAG CTG CTG TCT TTC ACT CGT-3¢. qPCR mix consisted of 0.5 mL DNA, 10 pmol of each primer, and 1• KiCStart SybrGreen qPCR ReadyMix (Sigma-Aldrich Life Science) in a final volume of 25 mL. Samples were run in duplicate on a ABI 7300 (Perkin Elmer). The thermocycling program included an initial step of 2 min at 50°C, subsequently 10 min at 95°C, followed by a 40 time repeat two-step cycle involving 95°C for 15 s and 60°C for 1 min.
Primers directed to porcine S100C gene were used as a pig DNA control. Primer sequences were as follows: MSR forward 5¢-CCA TCG GCC ATT GTT TTC CTG TTC A-3¢, MSR reverse 5¢-CCT CTG TGC CCA CCT GCT CTC TAC A-3¢, S100C forward 5¢-ATG CTG GAA GGG ACG GTA ACA ACA-3¢, and S100C reverse 5¢-GCT CAG CTG CTG TCT TTC ACT CGT-3¢. qPCR mix consisted of 0.5 mL DNA, 10 pmol of each primer, and 1• KiCStart SybrGreen qPCR ReadyMix (Sigma-Aldrich Life Science) in a final volume of 25 mL. Samples were run in duplicate on a ABI 7300 (Perkin Elmer). The thermocycling program included an initial step of 2 min at 50°C, subsequently 10 min at 95°C, followed by a 40 time repeat two-step cycle involving 95°C for 15 s and 60°C for 1 min.
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