Seven pairs of published oligonucleotide primers were used to detect the virulence genes using PCR. The individual PCRs, for each virulent gene, were set up in a 25 µL, which consisted of 12.5 µL AmpliTaq Gold® 360 PCR Master Mix (AmpliTaq Gold® DNA Polymerase 0.05 units/µL, Gold buffer 930 mM Tris/HCl pH 8.05, 100 mM KClO, 400 mM of each dNTP and 5 mM MgCl2) (Applied Biosystems, California, US). Then, 2.5 mM of each primer, 2 µL of template DNA and ddH2O were added to make the final volume. Test DNA was replaced with 5 µL of sterile nuclease-free water as negative control. Cycling conditions for PCR as well as the information of the published primers used are detailed in Table 1 .
Amplitaq gold 360 pcr master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
AmpliTaq Gold® 360 PCR Master Mix is a ready-to-use solution for performing polymerase chain reaction (PCR) amplification. It contains DNA polymerase, dNTPs, and PCR buffer components optimized for efficient and reliable DNA amplification.
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Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (6 mentions)
Abi 3500 genetic analyzer, by Thermo Fisher Scientific (5 mentions)
Sequencher 4, by Gene Codes (2 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Abi prism 3700 dna analyzer, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Gel extraction protocol, by Qiagen (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Abi prism 310, by Thermo Fisher Scientific (1 mentions)
Styi hf, by New England Biolabs (1 mentions)
Abi 3500 genetic analyser, by Thermo Fisher Scientific (1 mentions)
Qubit 3.0 fluorimeter, by Thermo Fisher Scientific (1 mentions)
Qubit dna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr cleanup kit, by Qiagen (1 mentions)
Bigdye terminator v3.1 sequencing kit, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer dna1000 kit, by Agilent Technologies (1 mentions)
21 protocols using amplitaq gold 360 pcr master mix
1
Virulence Gene Detection via Oligonucleotide PCR
2
Confirming PSEN1 H163Y Mutation
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To confirm the H163Y mutation in PSEN1, exon 6 was sequenced [25 (link)]. DNA was amplified using AmpliTaq Gold® 360 PCR Master Mix (Applied Biosystems). Primer sequences and PCR conditions are available upon request. The BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) was used for Sanger sequencing. Exon 6 in PSEN1 was sequenced in both directions and analyzed on an ABI 3500 Genetic Analyzer (Applied Biosystems).
For both brothers A and B, multiple DNA samples extracted on separate occasions from both blood (two different sample years) and skin biopsies (two different sample dates) were sequenced using three different methods: Sanger sequencing, next-generation gene panel sequencing, and whole-genome sequencing. Finally, cDNA extracted from fibroblast cultures was sequenced and confirmed the presence of both the wild-type transcript and the H163Y mutation transcript.
For both brothers A and B, multiple DNA samples extracted on separate occasions from both blood (two different sample years) and skin biopsies (two different sample dates) were sequenced using three different methods: Sanger sequencing, next-generation gene panel sequencing, and whole-genome sequencing. Finally, cDNA extracted from fibroblast cultures was sequenced and confirmed the presence of both the wild-type transcript and the H163Y mutation transcript.
3
Genomic DNA Extraction and Amplification
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Mutations in the tissues were determined as it has been described before 24 . Total DNA was extracted using proteinase K treatment followed by affinity‐purification using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer's instructions and the concentration of the DNA samples was measured by NanoDrop ND‐1000(Thermo scientific, Wilmington, DE). Genomic DNA (20–100 ng) was amplified in a reaction volume of 25 μL containing 12.5 μL AmpliTaq Gold® 360 PCR Master Mix (Applied Biosystems, Foster City, CA). Primer sequences used are listed in Table 2 . The PCR products were purified using Multiscreen‐HTS PCF Filter Plates (Merck, NJ) and then analyzed using ABI PRISM 3700 DNA analyzer (LifeTechnologies, Foster City, CA).
4
Screening for APP and PSEN1 Mutations
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Exons 16 and 17 in APP were sequenced to screen for the KM670/671NL and the E693G mutations [12 (link), 14 (link)]. To confirm the H163Y mutation in PSEN1, exon 6 was sequenced [26 (link)]. DNA was amplified using AmpliTaq Gold® 360 PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Primer sequences and PCR conditions are available upon request. The Big Dye® terminator v3.1 Cycle sequencing Kit (Applied Biosystems, Austin, TX, USA) was used for Sanger sequencing. The exons in APP and PSEN1 were sequenced in both directions and analyzed on an ABI3500 Genetic Analyzer (Applied Biosystems, Foster City).
5
Screening for APP and PSEN1 Mutations
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Exons 16 and 17 in APP were sequenced to screen for the KM670/671NL [54 (link)] and the E693G mutations [55 (link)]. To confirm the H163Y mutation in PSEN1 exon 6 was sequenced [56 (link)]. DNA was amplified using AmpliTaq Gold® 360 PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Primer sequences and PCR conditions are available upon request. Big Dye® terminator v3.1 Cycle sequencing Kit (Applied Biosystems, Austin, TX, USA) was used for Sanger sequencing. The exons in APP and PSEN1 were sequenced in both directions and analyzed on an ABI3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
6
Detecting DMD Gene Deletion Breakpoint
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PCR primer pairs (forward: 5'-GCTGTGGGTGAAAATGCCTT-3' and reverse:
5'-TGAAGGGACATTGGAGATTG-3') were used to amplify the region containing the breakpoint
between exon 44 and exon 51 caused by a deletion in the DMD gene.
Each PCR reaction contained AmpliTaq Gold® 360 PCR Master
Mix (Applied Biosystems), 10 μM primers and 50-100 ng of template gDNA/μL in a final
volume of 25 μL. The cycling conditions were: 95°C 10 min, 35 cycles of 95 °C for 15
s, 62 °C for 3 s and 72 °C for 60 s, and a final extension at 72 °C for 7 min. The
quality of the amplified products was assessed using agarose gel electrophoresis and
the PCR fragment was extracted from the gel using a Qiagen Gel extraction protocol
(QIAquick® gel extraction kit; Qiagen, Valencia, CA). Clean PCR product
was used for Sanger sequencing in a 3500 Series Genetic Analyzer.
5'-TGAAGGGACATTGGAGATTG-3') were used to amplify the region containing the breakpoint
between exon 44 and exon 51 caused by a deletion in the DMD gene.
Each PCR reaction contained AmpliTaq Gold® 360 PCR Master
Mix (Applied Biosystems), 10 μM primers and 50-100 ng of template gDNA/μL in a final
volume of 25 μL. The cycling conditions were: 95°C 10 min, 35 cycles of 95 °C for 15
s, 62 °C for 3 s and 72 °C for 60 s, and a final extension at 72 °C for 7 min. The
quality of the amplified products was assessed using agarose gel electrophoresis and
the PCR fragment was extracted from the gel using a Qiagen Gel extraction protocol
(QIAquick® gel extraction kit; Qiagen, Valencia, CA). Clean PCR product
was used for Sanger sequencing in a 3500 Series Genetic Analyzer.
7
Genetic Analysis of KRAS and BRAF
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KRAS and BRAF were amplified using polymerase chain reaction (PCR) with forward and reverse primers (see Supplemental Table 1, Supplementary Digital Content 1, http://links.lww.com/CTG/A718 ). Each 50 μL PCR reaction contained 100 nM of each primer, 1 ng template DNA, and master mix reagent (AmpliTaq Gold 360 PCR Master Mix; Applied Biosystems, Foster City, CA). Amplification conditions consisted of 10 minutes at 94 C, followed by 40 cycles at 94 C for 10 seconds, 55 C for 30 seconds, and 72 C for 30 seconds, in a thermal cycler (GeneAmp PCR System 9700; Applied Biosystems). The PCR products were separated by electrophoresis on 2% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. Then, the PCR products were purified before direct sequencing was performed using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems) on an ABI Prism 310 genetic analyzer (Applied Biosystems). KRAS mutations in codons 12 and 13 and BRAF mutation in codon 600 were examined according to the raw nucleotide sequencing data in waveform obtained by direct sequencing.
8
Molecular Identification of Tabanid Flies
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In order to supplement morphological identification of tabanid flies, PCR targeting the CO1 gene was conducted to identify tabanid fly DNA to species level and to further determine their phylogenetic position in comparison to other related taxa. The primers which amplified approximately 653 bp fragment of CO1 gene are shown in Table 1. In a total volume of 25 µl the PCR mixture contained 12.5 µl AmpliTaq Gold® 360 PCR Master Mix (AmpliTaq Gold® DNA Polymerase 0.05 units/ µl, Gold buffer [30 mM Tris/HCl pH 8.05, 100 mM KCl], 400 mM of each dNTP and 5 mM MgCl 2 ) (Applied Biosystems, California, USA), 2.5 mM of each primer, 2 µl of template DNA and double distilled water (ddH 2 O) was added to final volume. Genomic DNA of Glossina morsitans morsitans obtained from a colony of National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine was used as a positive control and double distilled water (ddH 2 O) as a no DNA template negative control. PCR conditions were set as described by Sari et al. (2012) .
9
PCR Amplification and Gel Electrophoresis
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The primers were designed by ArrayDesigner software. DNA amplifications were performed using AmpliTaq Gold® 360 PCR Master Mix (Thermo Fisher Scientific Inc.) and Mastercycler gradient thermal cycler supplied by Eppendorf at 35 cycles of denaturation at 95 °C for 1 min, primer annealing at 59 °C for 1 min, chain elongation at 72 °C for 2 min. After amplification 10 μl of PCR products were separated on 3% agarose gel and stained with ethidium bromide.
10
Genotyping Quail Mutation via PCR-RFLP
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The site of the nonsense mutation can be recognized by StyI in the L strain, but not in the WE strain (Fig. S8 ). By using this difference, we performed PCR-RFLP analysis to determine genotypes at the site of mutation in F2 offspring. Besides F2 offspring, 10 quail strains, including L, WE, AMRP, Quv, RWN, rb-TKP, NIES-FR/French, W (Wild), JW, and Estonian, were analyzed51 (link)–53 (link). We examined four individuals of each strain using genomic DNA that was extracted from whole blood. PCR was performed using a 25-μl reaction volume containing quail genomic DNA, 1 × AmpliTaq Gold® 360 PCR Master Mix (Thermo Fisher Scientific), and 2 μM of each primer. Primer sequences and PCR conditions are described in Table S8 . After digestion in a 20 μl reaction mixture containing 5 μl of PCR product, 10 units of StyI-HF (New England Biolabs), and 1 × CutSmart Buffer for 6 h at 37 °C, PCR products were electrophoresed in 2% agarose gel, followed by visualization with ethidium bromide.
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