DNA was isolated from peripheral blood cells using the QIAmp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) and subsequently amplified by standardized multiplex polymerase chain reaction (PCR) on the Thermalcycler (BIOMETRA, Göttingen, Germany). The Tyr2561 VWF-variant (VWF-V) fragment was identified using the 3130 Genetic Analyser (LIFE TECHNOLOGIES, Carlsbad, USA). In addition to the VWF-V we also tested for FVL and FIIV using the same multiplex PCR and fragment analysis.
3130 genetic analyser
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The 3130 Genetic Analyzer is a capillary electrophoresis instrument designed for DNA sequencing and fragment analysis. It utilizes four-color fluorescence detection to provide high-quality data output. The system features up to 16 capillaries and supports a range of applications, including Sanger sequencing, microsatellite analysis, and SNP genotyping.
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Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (8 mentions)
Qiaquick gel extraction kit, by Qiagen (3 mentions)
Abi prism bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (3 mentions)
Nzygelpure kit, by NZYTech (2 mentions)
Hi di formamide, by Thermo Fisher Scientific (2 mentions)
Thermal cycler, by Analytik Jena (1 mentions)
Qiamp dna blood mini kit, by Qiagen (1 mentions)
Genemapper id v3, by Thermo Fisher Scientific (1 mentions)
2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick dna kit, by Qiagen (1 mentions)
Genescan 500 rox size standard, by Thermo Fisher Scientific (1 mentions)
Ez1 robot, by Qiagen (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Quantitect probe master mix, by Qiagen (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
30 protocols using 3130 genetic analyser
1
Genetic Variant Analysis of VWF, FVL, and FIIV
2
Direct PCR for Fabric Samples
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Samples deposited on fabric, including toilet paper and tissue, were collected for direct PCR by taking two to three fibres, approximately 2-3mm in length, from the substrate and adding directly to the PCR reagents. NSF samples were added at 8 µL to the PCR and the total pellet from the SF was added, minus the sample removed for sperm scoring.
All samples were amplified without extraction using PowerPlex ESX 16 and 17 Kits (Promega Corp.). All samples were processed according to manufacturer's specifications; reaction volumes were reduced to 12.5 µL for samples processed with ESX 17. Samples treated with the differential enrichment protocol additionally had 1 x Amp Solution (Promega) added to the mastermix. For solid substrate samples (fibres, tissue and toilet paper) amplification-grade water was added up to the final volume required by the PCR kit.
Amplification took place in a 2720 Thermal Cycler (Life Technologies) and all batches were processed with both a negative and positive controls. All PCR products were visualised on a 3130 Genetic Analyser (Life Technologies, UK). Data were analysed using Genemapper ID v.3.2.1 software (Life Technologies, UK) with a threshold of 50 rfu used as the limit of detection, and 150 rfu as the limit for a homozygote. Local and global balance were calculated according to [41] .
All samples were amplified without extraction using PowerPlex ESX 16 and 17 Kits (Promega Corp.). All samples were processed according to manufacturer's specifications; reaction volumes were reduced to 12.5 µL for samples processed with ESX 17. Samples treated with the differential enrichment protocol additionally had 1 x Amp Solution (Promega) added to the mastermix. For solid substrate samples (fibres, tissue and toilet paper) amplification-grade water was added up to the final volume required by the PCR kit.
Amplification took place in a 2720 Thermal Cycler (Life Technologies) and all batches were processed with both a negative and positive controls. All PCR products were visualised on a 3130 Genetic Analyser (Life Technologies, UK). Data were analysed using Genemapper ID v.3.2.1 software (Life Technologies, UK) with a threshold of 50 rfu used as the limit of detection, and 150 rfu as the limit for a homozygote. Local and global balance were calculated according to [41] .
3
Molecular Characterization of MRSA and MRSE
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Methicillin resistant S. aureus and S. epidermidis isolates were submitted to molecular analysis and genomic DNA extraction was performed using the PureLink genomic DNA mini Kit (Invitrogen, CA, USA), and polymerase chain reaction was used to amplify the mecA gene, according to Jonas et al. [18 ]. Seven multilocus sequence typing (MLST) target genes were amplified using PCR according to the methods published for S. epidermidis and S. aureus available on the mlst.net webpage. The amplicons were sequenced with a 3130 Genetic Analyser (ABI 3130, Applied Biosystems, USA). The sequences were analysed using the Geneious software version 10.0.10 (Biomatters, New Zealand). Alleles and STs (sequencing types) were determined using the S. epidermidis (https://pubmlst.org/sepidermidis/ ) and S. aureus (http://saureus.beta.mlst.net/ ) MLST databases.
4
Genomic DNA Extraction and NR5A1 Sequencing
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Genomic DNA was extracted from peripheral blood leukocytes using the Qiaquick DNA kit (Qiagen, Hilden, Germany). Exons 1–7 of NR5A1 including the exon-intron boundaries were amplified and sequenced by direct cycle sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3130 Genetic Analyser (Applied Biosystems).
5
Multiplex PCR for MSI Analysis
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A multiplex PCR protocol was applied for MSI analysis. Samples for PCR were set up using the 2× Qiagen Multiplex PCR Kit to amplify five mononucleotide markers which were fluorescently labeled: BAT25(c-kit), BAT26(MSH2), NR21(SLC7A8), NR24(ZNF-2) and MONO27(MAP4K3) [6 (link)]. One microlitre of the template DNA extracted from formalin fixed tissue using the EZ1 Robot (Qiagen) was added to 25 ul PCR reaction. The following PCR programme was used to amplify selected markers: (Heated lid at 110 °C) 95 °C for 10 min, then 34 cycles of 94 °C for 1 min, 58 °C for 1 min and 72 °C 1 min, followed by 72 °C for 10 min. Following amplification, 1 ul of the PCR products were added to 0.5 ul of GeneScan™ 500 ROX™ Size Standard (an internal lane size standard for the Applied Biosystems fluorescence-based DNA electrophoresis systems) and 8.5 ul of formamide. Following denaturation (95 °C for 2 min and snap cooled on ice), the products were run on the 3130 genetic analyser (Applied Biosystems) and data processed using Genemarker software. Data supporting the conclusion of this article are included within the article.
6
Mapping Tiller Number Variation
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BC5 NIL76 was backcrossed to Banks to generate a large F2 mapping population consisting of several thousand F2 seed. DNA was extracted from the endosperm half of the F2 seed using the technique outlined by Ellis et al. (2005) (link). The SSR markers cfa2153 and psp2999 (Glu3-1A) that were flanking tin were used to identify recombinants. Corresponding F2 embryos were planted and progeny tested for tiller number in the F3 generation (at least 12 individuals per line). The Watkins lines were genotyped with the SSR marker gwm136 using capillary electrophoresis (Applied Biosystems 3130 Genetic Analyser) to resolve DNA fragment sizes.
7
Amplicon Sequencing Protocol
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Amplicons for sequencing were obtained using the primers and reaction conditions given in Table 2 . The PCR products were diluted 1:150 with distilled water prior to cycle sequencing. Cycle sequencing was performed with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, USA) in accordance with the manufacturer’s instructions using M13 primers. Cycle sequencing products were cleaned by an ethanol and ethylenediaminetetraacetic acid (EDTA) precipitation and sequenced on a 3130 Genetic Analyser (Applied Biosystems).
8
Genotyping of Viral VP-1/2 Gene by PCR and Sequencing
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For genotyping, the viral VP-1/2 gene was amplified using a conventional PCR assay. Briefly, 4 sets of forward and reverse primers (5’-CACAGACAGAAGCAGACGAGAT-3’ and 5’-GGTGAGAAGTGACAGCTGTATTG-3’ ; 5’-TTCAGAATGGTCACCTCTACA-3’ and 5’-CTGTGCTTCCGTTTTGTCTTA-3’ ; 5’-AACTTTGACTGTGAATGGGTTA-3’ and 5’-AAATAGTGCCTGGAGGATGAT-3’ ; 5’-CTATCACCAGAGAAAATCCAATC-3’ and 5’-GAGACGGTAACACCACTA-3’ ) were used in PCR amplification and the Quantitect Probe Master Mix (QIAGEN, Venlo, Netherlands) was used as the basis for the reaction mix. Viral products were analysed by electrophoresis on a 1.5% agarose gel and purified with the QIAquick Gel Extraction Kit (QIAGEN, Venlo, Netherlands). Sequencing reactions were set up with purified DNA, one of the specific primers used in the PCR and BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, California, USA) according to the protocol recommended by the manufacturer. Sequencing and sequence analysis were performed on a 3130 Genetic Analyser (Applied Biosystems, California, USA).
9
PCR Detection of EBV and CMV in Colon Tissues
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Detection of EBV and CMV in normal colon and tumor tissues was approached by conventional singleplex PCR in a 45-cycle reaction to improve the sensitivity of the assay [12 (link)]. PCR primers and conditions are shown in S1 Table . All the DNAs were tested in triplicate, and positive and negative DNA controls were included for each run. Analyses of TP53 and β-globin human genes were tested in parallel as controls for each DNA. A GeneAmp PCR System 9700 (Applied Biosystems) was used for PCR reactions, and electrophoresis to visualize amplification products in ethidium bromide-stained agarose gels. Positive results were considered when at least two of the three PCR replicates rendered a visible amplicon band with the expected size. To assess the specificity of the assay, 10 consecutive positive results for each virus were sequenced forward and reverse with a 3130 Genetic Analyser (Applied Biosystems), and the sequences’ results were aligned by BLASTn (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi ) for verification.
10
Identifying prey bacteria in Candidatus Uab amorphum
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Cells of ‘Ca. Uab amorphum’ and unidentified prey bacteria in the xenic culture were collected by centrifugation. Total DNA was extracted using the DNeasy plant mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PCR was performed using primer pairs specific for the bacterial 16S rRNA gene (27F and 1492R)45 ,46 (link). The PCR cycles (30 cycles) consisted of denaturation at 96 °C for 10 s, annealing at 55 °C for 30 min and extension at 68 °C for 2 min. The amplicon was purified with the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany), and was then cloned into the p-GEM T-easy vector (Promega, Tokyo, Japan). Ten clones were completely sequenced with a 3130 Genetic Analyser (Applied Biosystems, Foster City, CA), using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems). Sequenced clones were assembled into six sequences by the CodonCode Aligner (CodonCode Co., Centerville, MA) with a sequence similarity threshold of 99%. The 16-S rRNA gene sequence of ‘Ca. Uab amorphum’ was deposited in the GenBank database with accession code LC496071.
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