CSF (500 µl) was centrifuged at 12,000 × g for 1 min at 4°C, the supernatant was discarded and the precipitate was used for DNA extraction. To promote the efficient lysis of Gram-positive bacteria and fungi, and to enhance total DNA yields, lysozyme (100 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to the lysis buffer that was included in the Invitrogen PureLink Genomic DNA Mini kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total DNA was extracted with the Invitrogen PureLink Genomic DNA Mini kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The concentration of genomic DNA was quantified with the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Inc.). DNA quality and integrity were measured with an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) and an Agilent DNA 1000 kit (Agilent Technologies, Inc.), according to the manufacturer's protocol.
Invitrogen purelink genomic dna mini kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland
The Invitrogen PureLink Genomic DNA Mini kit is a DNA extraction and purification kit designed for the isolation of genomic DNA from a variety of sample types, including animal tissues, cells, and blood. The kit utilizes a spin-column based system to efficiently capture and purify genomic DNA.
Automatically generated - may contain errors
Lab products found in correlation
Lysozyme, by Merck Group (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Agilent dna 1000 kit, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions)
Sephadex g50, by Cytiva (1 mentions)
Abi prism 3100, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Masterpure yeast dna purification kit, by Illumina (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Proteinase k, by Roche (1 mentions)
3 protocols using invitrogen purelink genomic dna mini kit
1
CSF DNA Extraction and Quantification
2
Leech Mitochondrial and Nuclear Loci Amplification
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Tissue from the leech caudal sucker was removed and used for total DNA extraction using Invitrogen PureLink® Genomic DNA mini kit (ThermoFisher Scientific, Pittsburgh, Pennsylvania) following the manufacturer’s protocol. The next mitochondrial loci: cytochrome C oxidase subunit 1 (COI), nicotinamide adenine dinucleotide dehydrogenase subunit I (nad1), 12S rDNA and the nuclear: 18S rDNA, were amplified following Williams & Burreson (2006 (link)) and Saglam et al. (2018 (link)) protocols. PCR products were visualized by electrophoresis on an agarose gel. Successful amplifications were purified using CentriSep 96 filter plates (ThermoFisher Scientific, Pittsburgh, Pennsylvania) with Sephadex G-50 (Cytiva, Marlborough, Massachusetts). Sequencing reactions included 0.4 µl BigDye Terminator v. 3.1 (Applied Biosystems, Waltham, Massachusetts), 2 µl Buffer 5x, 4 µl ddH2O, 1 µl of primer at 10 µM, and 3 µl purified PCR product (total volume 10 µl). Samples were purified using Sephadex G-50, then 25 µl de EDTA 0.5 mM was added to each sample and finally sequenced in an ABI-PRISM 3100 (Applied Biosystems® Waltham, Massachusetts) sequencer at the Laboratorio Nacional de Biodiversidad (LANABIO), IB-UNAM. Complementary sequences were assembled and edited using Geneious ver. 5.1.7 (Biomatters Ltd., Auckland, New Zealand).
3
Fungal and Bacterial DNA Extraction from Swabs
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Swabs for fungal DNA extraction were added to extraction buffer (1 M of Tris–HCl (pH 8), 50 mmol/L of EDTA (Thermo Fisher Scientific, Rheinach, Switzerland), 0.5% Tween 20 (Bio‐Rad Laboratories, Cressier, Switzerland)) with proteinase K (Roche Diagnostics, Rotkreuz, Switzerland) and predigested at 56°C overnight. Swabs for bacterial DNA extraction were incubated in lysozyme digestion buffer (20 mg/mL lysozyme (Sigma‐Aldrich, Buchs, Switzerland), 25 mmol/L of Tris–HCl (pH 8), 2.5 mmol/L of EDTA (Thermo Fisher Scientific), 1% Triton X‐100 (Sigma‐Aldrich)) at 37°C for 60 min. After mechanical cell wall disruption with 0.5 mm beads (Qiagen, Hilden, Germany) in a Tissuelyser (Qiagen), samples were either processed with Masterpure Yeast DNA Purification kit (Epicentre, LuBioScience GmbH, Zurich, Switzerland) for fungal DNA or Invitrogen Purelink Genomic DNA Mini kit (Thermo Fisher Scientific) for bacterial DNA according to the manufacturers' protocols.
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