On the day of DNA isolation, duodenal aspirate microbial pellets under Allprotect were thawed on ice and 1x DTT was added in a 1:1 ratio and the mixture was vortexed.31 (link) Microbial DNAs were isolated from duodenal aspirates using the MagAttract PowerMicrobiome DNA/RNA EP Kit (Qiagen) on a KingFisher Duo (Thermo Fisher Scientific, Waltham, MA, USA).31 (link) Stool DNAs were isolated using MagAttract PowerSoil DNA EP Kits (Qiagen) and purified using a KingFisher Duo automated system (ThermoFisher Scientific, Waltham, MA). Isolated DNAs were quantified using Qubit 1X dsDNA, High Sensitivity Assay kits (Invitrogen by Thermo Fisher Scientific) on a Qubit 4 Fluorometer (Invitrogen, Carlsbad, CA, USA).
Kingfisher duo
Manufactured by Thermo Fisher Scientific
Sourced in United States
The KingFisher Duo is a compact, semi-automated magnetic particle processor designed for high-throughput nucleic acid extraction and purification. It utilizes magnetic separation technology to efficiently isolate and purify DNA, RNA, or proteins from a variety of sample types. The KingFisher Duo is a reliable and versatile tool for clinical, research, and diagnostic applications.
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Lab products found in correlation
Magattract powersoil dna kf kit, by Qiagen (8 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (6 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Qubit ds high sensitivity dna br assay kit, by Thermo Fisher Scientific (2 mentions)
Hiseq x, by Illumina (2 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (2 mentions)
Magattract powermicrobiome dna rna ep kit, by Qiagen (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Miseq sequencer, by Illumina (1 mentions)
Powermag soil dna isolation kit, by Qiagen (1 mentions)
Ez dna methylation kit, by Zymo Research (1 mentions)
Ez dna methylation kit, by Illumina (1 mentions)
Epic 850 k, by Illumina (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Prolong mounting medium, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Versant sample preparation kit 1, by Siemens (1 mentions)
Ncounter gene expression assay, by NanoString (1 mentions)
Nsolver software, by NanoString (1 mentions)
24 protocols using kingfisher duo
1
Duodenal Aspirate and Stool DNA Isolation
2
Efficient Protein Digestion and Quantification
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Protein samples were digested on-bead using multimode magnetic microparticles (MagReSyn® HILIC, ReSyn Biosciences) in a KingFisher Duo™ system (Thermo Fisher Scientific), as previously described [32 (link), 33 (link)], with minor modifications. Briefly, magnetic hydrophilic affinity microparticles (20 µl, 200 µg) were equilibrated in 200 µl 100 mM NH4Ac pH 4.5, 15% MeCN. The microparticles were transferred to a well containing the protein-binding buffer solution and mixed for 30 min at RT. The captured proteins were washed twice in 200 µl 95% MeCN and transferred to 200 µl 25 mM ammonium bicarbonate containing 1 µg sequencing grade modified trypsin (Promega, Madison, USA) and mixed for 4 h at 37 °C. Finally, beads were washed in 1% trifluoracetic acid to elute any remaining bound peptides. The resulting peptides (pool of digest and eluate) were vacuum dried, resuspended in 2% MeCN, 0.2% FA and quantified using the Pierce™ Peptide Quantification (Thermo Fisher Scientific) assay as per the manufacturer’s instructions.
A project specific system suitability-quality control (PQC) sample was prepared by pooling an equal volume of 16 urinary peptide samples. Additionally, a complex proteome digest was used as a general system suitability assessment. These PQC samples were injected at least once with each batch and analysed and processed together with the study samples.
A project specific system suitability-quality control (PQC) sample was prepared by pooling an equal volume of 16 urinary peptide samples. Additionally, a complex proteome digest was used as a general system suitability assessment. These PQC samples were injected at least once with each batch and analysed and processed together with the study samples.
3
Automated DNA Extraction and Purification
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DNA extraction was performed using the MagAttract PowerSoil DNA KF Kit (Qiagen) with some modifications as described previously (19 (link)). Extracted DNAs were purified using a KingFisher Duo automated system (Thermo Fisher Scientific, Waltham, MA), and DNA purity and concentration were determined using a NanoDrop One spectrophotometer (ThermoFisher Scientific).
4
Microbial Community DNA Extraction and Sequencing
5
Genome-wide DNA Methylation Analysis
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DNA was extracted from white blood cells of SKIPOGH participants using a bead-based KingFisher Duo robot extraction system (ThermoFisher, Waltham, Massachusetts), with 1.2 µg of DNA then treated with bisulfite using the EZ DNA Methylation© Kit (Zymo Research). Alternative incubation conditions (described in the EZ DNA Methylation™ Kit bisulfite conversion protocol, point 6 of the appendix) were used when performing the polymerase chain reaction (PCR) using the Illumina Infinium© Methylation Assay, and the final elution was carried out using 8 µl of M-Elution Buffer. DNA methylation levels were then assessed using genome-wide DNA methylation micro-array platforms Illumina HumanBeadChip 450 K (HM450K) (N = 250) and EPIC 850 K (EPIC) (N = 480). Missing CpG data were imputed using the nearest averaging multiple imputation method, beta coefficients were then logit transformed [4 , 5 (link)]. The CPACOR pipeline was used to normalize data and identify quality control issues [6 (link)]. Samples with issues identified in a quality control step, e.g., a call rate of less than 95% were excluded from all analyses (p value < 10–16) (HM450k: N = 9; EPIC: N = 15). CpGs identified as being present across both the EPIC and HM450k arrays (N = 452,453) were subsequently used for all analyses and calculations of epigenetic signatures.
6
Microbial DNA Extraction from Stool Samples
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Approximately 0.1 g of each stool sample was homogenized in 200 ml of sterile 1X PBS in a 2 ml sterile tube, and microbial DNA was isolated from each homogenate using the MagAttract PowerSoil DNA KF Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol with a few modifications. The lysis step was carried out by adding garnet beads and lysis buffer to each sample, followed by mechanical disruption for 10 min and high-speed centrifugation for 10 min. DNA was purified from the resulting supernatant on a KingFisher Duo (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s protocol. DNAs were quantified on a NanoDrop One™ Spectrophotometer (Thermo Fisher Scientific) and diluted to a concentration of 5 ng/ml for use in 16S rRNA sequencing.
7
Duodenal Microbiota DNA Extraction Protocol
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The duodenal aspirate pellets were thawed on ice and processed as previously described34 (link). Briefly, microbial DNA was isolated from each sample using the MagAttract PowerSoil DNA KF Kit (Qiagen) on a KingFisher Duo (Thermo Fisher Scientific, Waltham, MA, USA)34 (link). DNA quantification was carried out using the Qubit™ ds high sensitivity DNA BR Assay kit (Invitrogen by Thermo Fisher Scientific) on a Qubit™ 4 Fluorometer (Invitrogen, Carlsbad, CA, USA).
8
Identification of OSTF1-Interacting Proteins
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To identify OSTF1-interacting proteins, HEK293 cells cultured in DMEM/10% FCS/Pen-Strep were transiently transfected in triplicate with either empty vector (pCDNA3.1 V5-His) or V5-OSTF1 plasmids. After 48 h, cells were lysed in IP lysis buffer (50 mM Tris pH 7.5, 1% Triton-X100, 150 mM NaCl), protein content measured and subjected to automated IP on a Kingfisher Duo (Thermo) using anti-V5 magnetic agarose (MBL). Digestion and protein identification was performed by the MRC Institute of Genetics & Molecular Medicine Proteomics Facility.
9
Aptamer Selection for Lysozyme Detection
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The selection of aptamers with an affinity towards lysozyme (from hen’s egg-white) was performed by Just-in-Time-Selection in a buffer mimicking white wine conditions (1.5 mM potassium chloride, 14.5 mM potassium acetate, 3.5 mM magnesium acetate, 1.5 mM calcium acetate, 1 mM sodium dihydrogenphosphate, 4 mM potassium dihydrogenphosphate, 11.5% (v/v) ethanol, pH 3.1)18 19 (link)20 (link). Lysozyme was immobilized on magnetic particles so that following steps can be accomplished with a semi-automated magnetic separator (KingFisher Duo, Thermo Fisher Scientific Oy, Vantaa, Finland). The consecutive amplification was performed using BEAMing (beads, emulsion, amplification, magnetic) to enable an amplification with a lower number of by-products. Furthermore, the use of this technique combines amplification and strand separation in a single step37 (link). After 15 turns, the resulting aptamers were sequenced and characterized regarding their affinity towards lysozyme by surface plasmon resonance (SPR). Based on the sequences obtained, fluorescently labelled aptamers were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
10
Keratinocyte Cell-Free DNA Extraction
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To get cell-free DNA, a tube containing a pellet of 300,000 keratinocyte cells was added to 400µL of Crime Lysis Buffer, 50µL of Proteinase K, and 4.5 µL of 3 M Dithiothreitol (Crime Prep, Adem-Kit AutoMag solution, Ademtech, France). The tube was placed in a thermomixer and incubated at + 56 °C at 500 rpm for 40 min. After 2 min of centrifugation at 3500 rpm, DNA was extracted with a KingFisher DUO (Thermofisher, USA) according to the manufacturer’s protocol.
To quantify the different DNA extracts, a NanoDrop One (Thermofisher, USA) was used. A 2 µl drop of the extract was deposited and measured. A concentration in ng/µl was then calculated.
To establish our strategy detection14 (link):
To quantify the different DNA extracts, a NanoDrop One (Thermofisher, USA) was used. A 2 µl drop of the extract was deposited and measured. A concentration in ng/µl was then calculated.
To establish our strategy detection14 (link):
11.5 ng of cell-free DNA was deposited on glass slides.
A mixture of keratinocyte cells and 11.5 ng of cell-free DNA were placed on glass slides.
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