PCR primer sets were designed to amplify several inter- and intragenic mitochondrial genes: cox2-partial gene, nad6, rpl5-ψrps14, rps1, rps4, and rps10 (Table S2 ). PCR amplification was carried out in a 10 μl reaction solution that included 2 μl of DNA (5 ng/μl), 5 μl of 2 × Ampdirect® Plus (Shimadzu, Japan), 0.25 U of heat-activated Taq DNA polymerase (LA Taq® Hot Start Polymerase, Takara Bio, Inc., Kusatsu, Japan), and forward and reverse primers (each at 0.3 μM). The thermal cycling profile was as follows: 95 °C for 10 min, followed by an initial cycle of 94 ℃ for 30 s, 65 ℃ for 30 s, and 72 ℃ for 1 min; a reduction in the annealing temperature by 1 ℃ during each of the next five cycles, followed by an annealing temperature of 60 ℃ for the remaining 25 cycles; and a final extension at 72 ℃ for 5 min. The amplification products were electrophoresed on a 2% agarose gel with 1 × TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA). The PCR products were subjected to Sanger-sequencing by a commercial company (Takara Bio, Inc., Kusatsu, Japan).
Ampdirect plus
Manufactured by Shimadzu
Sourced in Japan
The Ampdirect Plus is a laboratory equipment product from Shimadzu. It is designed to amplify and analyze samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Biotaq hs dna polymerase, by Meridian Bioscience (3 mentions)
La taq hot start polymerase, by Takara Bio (1 mentions)
Blend taq plus, by Toyobo (1 mentions)
Dneasy 96 plant kit, by Qiagen (1 mentions)
Ex taq dna polymerase, by Takara Bio (1 mentions)
Rox dye, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Agarose s, by Nippon Gene (1 mentions)
Gelred, by Biotium (1 mentions)
Kod plus ver 2, by Toyobo (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Fastgene gel pcr extraction kit, by Nippon Genetics (1 mentions)
7 protocols using ampdirect plus
1
Mitochondrial Gene Amplification and Sequencing
2
Genotyping Cucumis melo Genes
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Genomic DNA was isolated from young leaves of the parental lines and F 1 and F 2 populations using the DNeasy Plant Kit 96 (Qiagen, Hilden, Germany). CmACS-7 and CmWIP1 were genotyped according to Boualem et al. (2008) (link) and Chen et al. (2016) (link). Amplification was carried out in 10 µL containing 2 µL of total DNA (5 ng/µL), 5 µL of 2× Ampdirect Plus (Shimadzu, Kyoto, Japan), 0.1 µL of Blend Taq Plus (2.5 U/µL) (Toyobo, Osaka, Japan), and 10 µM of forward and reverse primers (Table S1). For CmWIP1 markers, the amplification products were electrophoresed in 2% agarose gel in 1× TAE buffer. For the CmACS-7 CAPS marker, the amplification products were digested with AluI (Takara Bio, Shiga, Japan) and separated in 2% agarose gel in 1× TAE buffer.
3
Quantitative PCR for Stool DNA Analysis
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qPCR was performed in 384-well optical plates on an ABI PRISM 7900HT Sequence Detection System (Life Technologies, Foster City, CA). Ampdirect Plus (Shimadzu, Kyoto, Japan), a commercial PCR buffer, was used to neutralize inhibitory factors in stool DNA templates. Each reaction mixture of 20 µL was composed of 0.4 units of ExTaq DNA polymerase (TaKaRa Bio Inc., Shiga, Japan), 10 µL of 2×Ampdirect Plus, 0.4 µL of Rox dye (Life Technologies), 0.2 µM of each specific primer, 0.2 µM of the fluorescent probe, and 5 µL of template DNA. The amplification program consisted of one cycle at 95°C for 30 s and then 50 cycles at 95°C for 5 s and 56°C for 50 s.
4
Molecular Confirmation of Tsetse Fly Species
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For molecular confirmation of the tsetse fly species, GlossinaITS1_for (5′-GTG ATC CAC CGC TTA GAG TGA−3′) and GlossinaITS1_rev (5′-GCA AAA GTT GAC CGA ACT TGA−3′) primers were used to amplify the ITS1 region of ribosomal genes of tsetse flies (46 (link)). Reactions contained 1–10 ng of template DNA, 1 × Ampdirect Plus (Shimadzu Corp., Kyoto, Japan), 0.25 U BioTaq HS DNA Polymerase (Bioline, Memphis, TN, USA), 0.2 mM primers, and distilled water to a total volume of 10 μL. Amplification included an initial denaturation step at 95°C for 10 min, followed by 30 cycles each of 94°C for 30 s, 62°C for 1 min, 72°C for 2 min, and a final extension step at 72°C for 7 min. The band patterns were visually inspected after gel electrophoresis (G. pallidipes: 920 bp, G. m. centralis: 800 bp and 150 bp). There was a total of 212 G. pallidipes and 86 G. m. centralis samples.
5
Molecular Identification of Potato Resistance Genes
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DNA was extracted from fresh leaves using the one-minute DNA extraction method (Hosaka 2004 ). The presence of four resistance genes (R1, Rx1, Rychc, and H1) were estimated using the following markers: R1-specific marker (76-2sf2: 5′-CACTCGTGACATATCCTCACTA-3′ and 76-2SR: 5′-CAACCCTGGCATGCCACG-3′, Ballvora et al. 2002 (link)), Rx1-linked marker (RxSP-S3: 5′-ATCTTGGTTTGAATACATGG-3′ and RxSP-A2: 5′-CACAATATTGGAAGGATTCA-3′, Ohbayashi and Komura 2004 ), Rychc-linked marker RAPD 38-530 (5′-TTCGAGCCAG-3′, Hosaka et al. 2001 ), and H1-linked marker (H1SP-S2: 5′-GAGCTCAGAGGTGAAAAATA-3′ and H1SP-A6: 5′-GGAACAATGTTGAATGCAAG-3′, Ohbayashi et al. 2010 ). Diagnostic marker bands were amplified using polymerase chain reaction (PCR) in a volume of 10 μl consisting of 2 μl of template DNA, 5 μl of 2× Ampdirect® Plus (Shimadzu Co., Japan), 0.25 units of Taq DNA polymerase (NovaTaqTM Hot Start DNA Polymerase, Novagen, USA), and 1 μl each of 3 μM forward and reverse primers. The thermal conditions for PCR were similar to those described in the original literature. The detection procedure for the RAPD marker 38-530 is described in Hosaka (2004) . The amplified product was separated on a 1.4% agarose gel in 1× TAE buffer (40 mM Tris-acetate and 1 mM EDTA).
6
Molecular Detection of T. b. rhodesiense
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SRA PCR was employed to identify T. b. rhodesiense using the primers described by Radwanska et al. [47 (link)] (Supplementary Table 2 ). Reagents used for each reaction included 5 μL Ampdirect plus (Shimadzu, Japan), 0.05 μL BIOTAQ HS DNA Polymerase (5 U/μL) (Bioline, UK), 0.2 μL of each 10 mM primer, 2.55 μL RNase-free water, and 2 μL extracted DNA. The temperature and cycling profile included an initial hold for 10 min at 95 °C, followed by 40 cycles at 94 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 5 min. PCR products were examined by electrophoresis in 2% agarose S (Nippongene, Japan) in TAE buffer and stained using GelRed (Biotium, USA) dye before being visualized under ultraviolet light.
7
Mitochondrial COI Amplification and Sequencing
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The mitochondrial COI was amplified using the primers LCO 1490 (5'-GGTCAACAAATCATAAAGATATTGG3') and HCO 2198
(5'TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al., 1994) . The reaction was carried out in a volume of 15 µl using a pair of primers (0.4 µM each), Ampdirect Plus (Shimadzu Biotech, Tsukuba, Japan), and BIOTaq HS DNA polymerase (Bioline, London, UK). After an initial denaturation at 95 °C for 5 min, amplification was performed with 35 cycles consisting of denaturation at 95 °C for 1 min, annealing at 49 °C for 30 sec, extension at 72 °C for 2 min, followed by a final extension at 72 °C for 10 min. PCR products were purified using a FastGene
Gel/PCR Extraction kit (NIPPON Genetics) and sequenced with a forward primer by the dideoxy chain termination method using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). To confirm the authenticity of the COI sequences obtained, some individuals from each sand fly species (in total 40 specimens) were subjected to PCR using a high fidelity DNA polymerase (KOD-Plus-ver.2; TOYOBO, Tokyo, Japan), and the sequences were determined as described above.
(5'TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al., 1994) . The reaction was carried out in a volume of 15 µl using a pair of primers (0.4 µM each), Ampdirect Plus (Shimadzu Biotech, Tsukuba, Japan), and BIOTaq HS DNA polymerase (Bioline, London, UK). After an initial denaturation at 95 °C for 5 min, amplification was performed with 35 cycles consisting of denaturation at 95 °C for 1 min, annealing at 49 °C for 30 sec, extension at 72 °C for 2 min, followed by a final extension at 72 °C for 10 min. PCR products were purified using a FastGene
Gel/PCR Extraction kit (NIPPON Genetics) and sequenced with a forward primer by the dideoxy chain termination method using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). To confirm the authenticity of the COI sequences obtained, some individuals from each sand fly species (in total 40 specimens) were subjected to PCR using a high fidelity DNA polymerase (KOD-Plus-ver.2; TOYOBO, Tokyo, Japan), and the sequences were determined as described above.
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