Genomic DNA was extracted from blood leukocytes using the KingFisher™ Duo Prime Purification System (Thermo Scientific™) according to the manufacturer’s protocol. Genotyping of the SNPs DRD2/ANKK1 TaqIA (NCBI accession number: rs1800497) and COMT Val108/158Met (rs4680) was performed using PCR-based restriction fragment length analysis according to previously described protocols (Schott et al. 2006 (link); Wimber et al. 2011 (link); Richter et al. 2013 (link), 2014 (link), 2017 (link)). A1 carriers of the TaqIA SNP were grouped together (A1 + : A1/A1 and A1/A2; A1 − : A2/A2) as in the previous studies (Klein et al. 2007 (link); Frank and Hutchison, 2009 (link); Jocham et al. 2009 (link); Stelzel et al. 2010 (link); Stice et al. 2012 (link); Richter et al. 2013 (link), 2014 (link), 2017 (link)).
Kingfisher duo prime purification system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The KingFisher Duo Prime Purification System is a compact, automated instrument designed for the purification of nucleic acids and proteins. The system utilizes magnetic particle-based separation technology to efficiently extract and purify target molecules from a variety of sample types.
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Lab products found in correlation
Mag bind cfdna kit, by Omega Bio-Tek (2 mentions)
Magmax core nucleic acid purification kit, by Thermo Fisher Scientific (2 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Membrane filter, by Merck Group (1 mentions)
Magmax pathogen rna dna kit, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Magmax ffpe dna rna ultra kit, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Cobas 6800 system, by Roche (1 mentions)
Cobas sars cov 2 test, by Roche (1 mentions)
Allplex sars cov 2 assay, by Seegene (1 mentions)
Qiasymphony dsp virus pathogen mini kit, by Qiagen (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Magmax mirvana total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mag bind viral dna rna 96 kit, by Omega Bio-Tek (1 mentions)
Rnalater stabilization solution, by Thermo Fisher Scientific (1 mentions)
Fisherbrand commode specimen collection system, by Thermo Fisher Scientific (1 mentions)
20 protocols using kingfisher duo prime purification system
1
Genotyping of DRD2/ANKK1 and COMT Polymorphisms
2
Magnetic Bead-Based cfDNA Extraction
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Samples for cfDNA enrichment were loaded into the King Fisher Duo Prime Purification system (Thermo Fisher Scientific, Waltham, Massachusetts, USA) for magnetic beads DNA extraction following centrifugation to prevent possible gDNA leakage from cell residues. The Mag-Bind cfDNA Kit (Omega Bio-tek, Georgia, USA) was used for extraction of cfDNA from plasma and urine following the recommendations of the producer. Different starting volumes of samples were tested, ranging from 100–2,000 μl. For processing porcine urine, 1,000 μl samples were used and eluted into a final volume of 200 μl. Once the samples were eluted into buffer, they were stored for up to 12 h at 4°C, before they were determined by qRT-PCR and fluorescence quantification.
3
Genomic DNA Extraction and GBS Library Preparation
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Whole-cell genomic DNA was extracted using the KingFisher™ Duo Prime Purification System (Thermo Fisher Scientific Inc., Waltham, USA) following the manufacturer’s protocol for the KingFisher™ Cell and Tissue DNA purification kit (Thermo Fisher Scientific Inc., Waltham, USA). The DNA concentration was estimated using a Qubit™ 2.0 fluorometer (Thermo Fisher Scientific Inc., Waltham, USA). The fragment size range of DNA extractions was estimated for a subset of DNA extractions using an Agilent 4200 TapeStation™ (Agilent Inc.). DNA extractions were subsequently cleaned using the ZR-96 Genomic DNA Clean & Concentrator™ (Zymo Research Inc., Orange, CA, USA) following the manufacturer’s protocol. Population genomic data were generated from the DNA extracts following the GBS approach originally developed by Elshire et al. [33 (link)] at the Institute of Biotechnology commercial service (Cornell University, NY, USA) following their standard pipeline [33 (link)]. The genomic DNA extracts were digested with the DNA restriction endonuclease EcoT221, which has a six-base pair (bp) recognition sequence, and fragments in the size range from 200 to 380 bps were used as the basis for the GBS libraries.
4
Automated DNA Extraction from Blood
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Genomic DNA was extracted from 200 μL of blood pellet samples with the bead-based automated kit MagMAX™CORE Nucleic Acid Purification Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's recommendations. The extraction was processed using the KingFisher™ Duo Prime Purification System (ThermoFisher Scientific, Pittsburgh, PA, USA). Genomic material was eluted in 90 μL of elution buffer and stored at −20 °C until required.
5
Nucleic Acid Extraction from Cell Lines
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Nucleic acid was extracted from both tissue homogenate and supernatant from cell lines with an observed cytopathic effect (CPE). The flask containing BF2cell lines where 75% of the surface showed CPE development was frozen at −80 °C overnight, thawed, centrifuged, and the supernatant was filtered through a membrane filter (0.45 μm; Millipore, Germany), aliquoted and refrozen at −80 °C. DNA extraction and purification were performed from 200 µL filtrate and 200 µL tissue homogenate using a MagMAX CORE Nucleic Acid Purification Kit for rapid purification of high-quality DNA and RNA for downstream molecular analysis (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s protocol on a KingFisher Duo Prime Purification System (Thermo Scientific, Waltham, MA, USA).
6
Optimized cfDNA Extraction from Plasma
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CfDNA was initially extracted from plasma using the Mag-Bind cfDNA Kit (Omega Biotek), following the recommendations of the KingFisher Duo Prime Purification System (Thermo Fisher Scientific). Due to the low input starting volume of 450 μl, some modifications to the protocol were necessary to ensure high enough cfDNA concentrations for down-stream applications. Collect count in the elution step was increased from three to four and the final elution volume was set to 80 μl. Phosphate-buffered saline (PBS) was added to three of the samples (piglet nr. 21 at baseline and piglet nr. 20 and nr. 25 at the end of hypoxia) to equalize the settings of the starting plasma volume for cfDNA extraction. Samples of extracted cfDNA were stored at − 80 °C for up to one week until being further processed.
7
Fecal RNA/DNA Extraction Protocol
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Both RNA and DNA were extracted from 200 µL of fecal samples using 5X MagMax Pathogen RNA/DNA Kit (Thermo Fisher Scientific, Illkich, France) by using the fecal samples preserved in 0.5 mL of home-made preservative RNA solution. The feces were vortexed vigorously (30 Hz) using a Retsch MM400 Tissue lyser for 5 min to fully homogenize and mix well the fecal particles, followed by centrifugation at 16,000× g for ~3 min to fully separate the supernatant from the fecal pellet. A total of 130 µL of the supernatant was collected to isolate and purify the nucleic acids using a Mag Max extraction kit (Thermo Fisher Scientific, France) with the automatic KingFisher Duo Prime Purification System (Thermo Fisher Scientific, France) following the manufacturer’s instructions. A final volume of 80 µL of eluted RNA/DNA was collected and stored at −80 °C.
8
Nucleic Acid Extraction from Serum Samples
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Nucleic acid was extracted from serum samples (n = 27) using the KingFisher Duo Prime Purification System (ThermoFisher Scientific, Waltham, MA, USA) and the IndiMag Pathogen Kit (Indical Bioscience, Leipzig, Germany). To exclude false-negative results caused by PCR inhibitors and to verify RNA extraction and amplification, an RNA template control (intype IC-RNA, Indical Bioscience) was added to each sample. Due to negative internal controls, the RT-PCRs could not be evaluated for serum samples from two adult males, one captured in Wichebukta (9; DL 99/10; 2001) and the other in Tempelfjorden (27; DL 16/05; 2016) (Table 2 ).
9
Immunoprecipitation of FLAG-KRAS Proteins
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Caco-2 cell lysates expressing FLAG-KRAS proteins were immunoprecipitated from 800 μg of cell lysate using anti-FLAG-M2 magnetic beads (M8823; Sigma-Aldrich) using the KingFisher DuoPrime purification system (Thermo Fisher Scientific). Beads were washed in TBS (according to the manufacturer’s instructions) for 5 min, twice, at low speed. Then, beads were collected by the KingFisher magnet and discarded into the samples wells and mixed at a slow speed for 1 h. Beads–antibody–samples were collected and went through different wash salted solutions (Wash 1 and Wash 2: RIPA buffer with 150 mM NaCl; Wash 3: RIPA buffer with 500 mM NaCl), mixed at low speed for 30 s. Beads–antibody–samples were eluted in 50 μl of glycine (0.1 M, pH 3.0) for 5 min. Immediately after, samples were neutralized with 20 μl of Tris base (1 M, pH 8.0).
10
Dried Blood Spot DNA/RNA Extraction
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Capillary blood drops were collected on Whatman® protein saver cards (Sigma Aldrich, St. Louis, MO, USA), and are described as dried blood spots (DBS). Total DNA and RNA were isolated from DBS using MagMAX FFPE DNA/RNA ultra-kit via a KingFisher Duo Prime purification system (both Thermo Fisher Scientific, Waltham, MA, USA). A nanodrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA) was used for quantity and quality checks of DNA and RNA samples.
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