Genomic DNA was extracted from fingertip blood samples obtained from the graded exercise tests (GXT) using the GeneJET Genomic Whole Blood DNA Purification Kit (#K0781 Thermo Scientific, MA, USA). ACTN3 gene variants were determined in duplicate using the TaqMan SNP assay (Applied Biosystems, Thermo Fisher Scientific, CA, USA) by Mastercycler® ep realplex2 (Eppendorf, Hamburg, Germany). Genotyping was replicated in another independent institute (The Murdoch Childrens Research Institute, Melbourne), as previously described29 (link), to validate the results.
Genejet whole blood genomic dna purification kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
The GeneJet Whole Blood Genomic DNA Purification kit is a laboratory product designed to extract and purify genomic DNA from whole blood samples. The kit utilizes a silica-based membrane technology to efficiently capture and isolate DNA, resulting in high-quality genomic DNA samples suitable for downstream molecular biology applications.
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Lab products found in correlation
Taqman snp assay, by Thermo Fisher Scientific (2 mentions)
Tbe polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Gelred stain, by Biotium (2 mentions)
Realplex mastercycler 2, by Eppendorf (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Mastercycler ep realplex2, by Eppendorf (1 mentions)
Magsep blood gdna kit, by Eppendorf (1 mentions)
Quantstudio 7 flex, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Uv vis spectrophotometer, by Quawell (1 mentions)
Nanodrop, by Quawell (1 mentions)
Casava 1, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
12 protocols using genejet whole blood genomic dna purification kit
1
Genotyping of ACTN3 in Blood
2
Validating Genetic Variants Linked to Muscle and Bone
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We will target either candidate gene variants [48 (link),49 (link)] or Genome-Wide Association–based variants previously related to skeletal muscle and bone health [50 (link),51 (link)]. Genomic DNA will be extracted from residual blood samples from Becton Dickinson (BD) Vacutainer EDTA tubes using the MagSep Blood gDNA kit (0030 451.00, Eppendorf) or GeneJET Genomic Whole Blood DNA Purification Kit (#K0781 Thermo Scientific). Gene variants will be determined using the TaqMan SNP assay (Applied Biosystems, Thermo Fisher Scientific) by QuantStudio 7 Flex (Applied Biosystems, Thermo Fisher Scientific). Genotyping will be replicated in another independent institute, as previously described [52 (link),53 (link)], to validate the results.
3
Genotyping VDBP Alleles by RFLP
4
Genomic DNA Isolation from Animal Blood
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The blood samples collected from different animals species presented at Veterinary Clinical Complex (VCC), Veterinary College, Junagadh were used in the present investigation. About 2–3 ml jugular vein blood was collected from each animal in a vial containing EDTA, and various animal parameters like age, sex, breed, and species were recorded. During 2015–2020, 1250 samples were collected from cattle, buffaloes, horses, dogs, sheep, goats, and wild felids. Randomly, 468 samples were chosen for microscopic examination (Supplementary Table 1). The samples found positive in microscopy were used as the positive control. The samples collected from apparently healthy cow calves maintained in the institute farm were used as the negative control.
The whole blood genomic DNA was isolated from 200 µl of blood sample using GeneJET Whole Blood Genomic DNA Purification Kit (Thermo Scientific, Lithuania) following the manufacturer's protocol. Finally, DNA was eluted in 200 µl of elution buffer and stored at − 20 °C till further use.
The whole blood genomic DNA was isolated from 200 µl of blood sample using GeneJET Whole Blood Genomic DNA Purification Kit (Thermo Scientific, Lithuania) following the manufacturer's protocol. Finally, DNA was eluted in 200 µl of elution buffer and stored at − 20 °C till further use.
5
VDBP Allele Analysis by RFLP
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Three VDBP alleles were analyzed: Gc1F (rs7041-T/rs4588-C), Gc1S (rs7041-G/rs4588-C), and Gc2 (rs7041-T/rs4588-A).7 (link) DNA was extracted from blood using the GeneJet Whole Blood Genomic DNA Purification kit (Thermo Fisher, Waltham, MA, USA). Genotypes were then determined by restriction-fragment length polymorphism (RFLP) analyses, as previously described.21 (link) Briefly, DNA was amplified by PCR using a Realplex Mastercycler2 (Eppendorf, Hauppauge, NY, USA) and oligonucleotide primers: forward, 5′-TATGATCTCGAAGAGGCATG-3′; reverse, 5′-AATCACAGTAAAGAGGAGGT-3′ (synthesized by Integrated DNA Technologies, Coralville, IA, USA; GenBank L10641.1). PCR amplicons were then treated with either HaeIII or StyI restriction enzymes that cleave at Gc1S or Gc2 polymorphic sites, respectively. Resulting fragments were separated on 4–12% TBE polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and visualized by GelRed stain (Biotium, Fremont, CA, USA) to assign one of six possible genotypes based on fragment sizes. RFLP analyses were repeated for any samples with unclear results; also, random samples were repeated for quality control. PCR reagents and enzymes were from New England Biolabs (Ipswich, MA, USA).
6
Molecular Identification of Filarioid Species
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Molecular analysis was performed to differentiate between and accurately identify the filarioid species. DNA was isolated from 200 µl aliquots of EDTA blood using a GeneJet Whole Blood Genomic DNA Purification Kit (Thermo Fisher Scientific, Vilnius, Lithuania) according to the manufacturer’s instructions. DNA from mature helminths was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
To identify filarioid species, conventional PCR and pan-filarial primers (DIDR-F1, DIDR-R1) were used that amplify fragments of different length of the internal transcribed spacer region 2 (ITS2) of the ribosomal DNA from six different filarioid species (D. repens, D. immitis, Acanthocheilonema reconditum, Acanthocheilonema dracunculoides, Brugia pahangi and Brugia malayi) [23 (link)]. The PCR was carried out as described by Rishniw et al. [23 (link)]. The identification was performed based on 484 bp fragments for D. repens. Blood samples positive for microfilaria and adult nematode samples were then verified with a D. repens-specific primer set (DR COI-F1/DR COI-R1) based on partial (209 bp) amplification of cytochrome oxidase subunit 1 (cox1) gene, as described by Rishniw et al. [23 (link)].
To identify filarioid species, conventional PCR and pan-filarial primers (DIDR-F1, DIDR-R1) were used that amplify fragments of different length of the internal transcribed spacer region 2 (ITS2) of the ribosomal DNA from six different filarioid species (D. repens, D. immitis, Acanthocheilonema reconditum, Acanthocheilonema dracunculoides, Brugia pahangi and Brugia malayi) [23 (link)]. The PCR was carried out as described by Rishniw et al. [23 (link)]. The identification was performed based on 484 bp fragments for D. repens. Blood samples positive for microfilaria and adult nematode samples were then verified with a D. repens-specific primer set (DR COI-F1/DR COI-R1) based on partial (209 bp) amplification of cytochrome oxidase subunit 1 (cox1) gene, as described by Rishniw et al. [23 (link)].
7
Whole Blood DNA Extraction and SNP Analysis
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Genomic DNA was extracted from 3 mL EDTC-anticoagulated blood samples using a Gene JET Whole Blood Genomic DNA Purification kit (Thermo Scientific Co. Ltd.). SNPs were analyzed through multiplex PCR for targeted next-generation sequencing.
8
Genetic Diversity Assessment of Cholistani and Crossbred Cattle
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In this study, 50 and 48 male animals from each of the two breed
populations, viz. Cholistani and crossbred cattle, were taken at random from different areas in
the country following FAO guidelines on the selection animals and unrelatedness
(FAO, 2011). The crossbred animals were a cross between Cholistani and
Holstein Friesian with F2 or later generations. The pedigree information of
sampled animals was available in the records of the Livestock Experiment Station as
well as the Semen Production Unit Qadarabad and Karaniwala. Blood samples were
collected in sterile tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant and samples were
shipped to the Laboratories of Animal Breeding and Genetics, PMAS Arid
Agriculture University, Rawalpindi, for further analyses. Genomic DNA was
extracted from blood samples according to standard manufacturer's protocols
using the GeneJET Whole Blood Genomic DNA Purification Kit (Thermo Scientific).
The quality of the DNA extracted was tested with Quawell Nanodrop (Q 5000,
Quawell UV-VIS Spectrophotometer, USA), and DNA was kept at C
until further use in the study.
populations, viz. Cholistani and crossbred cattle, were taken at random from different areas in
the country following FAO guidelines on the selection animals and unrelatedness
(FAO, 2011). The crossbred animals were a cross between Cholistani and
Holstein Friesian with F2 or later generations. The pedigree information of
sampled animals was available in the records of the Livestock Experiment Station as
well as the Semen Production Unit Qadarabad and Karaniwala. Blood samples were
collected in sterile tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant and samples were
shipped to the Laboratories of Animal Breeding and Genetics, PMAS Arid
Agriculture University, Rawalpindi, for further analyses. Genomic DNA was
extracted from blood samples according to standard manufacturer's protocols
using the GeneJET Whole Blood Genomic DNA Purification Kit (Thermo Scientific).
The quality of the DNA extracted was tested with Quawell Nanodrop (Q 5000,
Quawell UV-VIS Spectrophotometer, USA), and DNA was kept at C
until further use in the study.
9
DNA Extraction and Sequencing Protocol
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Genomic DNA was extracted from blood with the GeneJET (Whole Blood) genomic DNA purification Kit (Thermo Scientific, USA), according to manufacturer's protocol. DNA Library preparation was carried out according to the manufacturer's instructions for sequencing on HiSeq2000 (Illumina, San Diego, CA, USA). For demultiplexing and conversion to FASTQ format, CASAVA 1.8.2 (Illumina) was used.
10
Genomic DNA Extraction from Canine Blood
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Genomic DNA was extracted from 200 μL aliquots of EDTA blood (taken from the vena cephalica of the examined dog) using the GeneJet Whole Blood Genomic DNA Purification kit (Thermo Fisher Scientific, Vilnius, Lithuania), according to the manufacturer’s instructions.
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