mtCOI DNA was amplified using the primer pair HCO-2198 and LCO-1490 [49 (link)]. The PCR process was conducted with a total reaction volume of 20 μL, which included 10 μL SmartGene 2× Dye Mixed Taq (SmartGENE, Daejeon, Republic of Korea), 1 μL of each primer (10 pmol/μL), 5 μL of nuclease-free water and 3 μL of template DNA solution (40 ng). The reaction mixtures underwent amplification with the following parameters: an initial denaturation at 94 °C for 5 min; this was followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 60 s; and a final extension at 72 °C for 5 min. This process was carried out in a T100 Thermal Cycler from Bio-Rad (Hercules, CA, USA). The PCR products were electrophoresed on a 1% agarose gel, which was then purified using ExpinTM Gel SV (GeneAll, Seoul, Republic of Korea). The PCR amplicons were subcloned into a cloning vector (the pGEM-T Easy vector, Promega, Madison, WI, USA) and sequenced using Sanger sequencing (Macrogen, Seoul, Republic of Korea).
Expin gel sv
Manufactured by GeneAll
Sourced in United States
The Expin Gel SV is a lab equipment product designed for the extraction and purification of DNA from agarose gels. It is a simple and efficient tool for isolating DNA fragments from electrophoresis gels.
Automatically generated - may contain errors
Lab products found in correlation
Psicheck 2 vector, by Promega (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Exprep plasmid sv mini, by GeneAll (2 mentions)
Ligafast rapid dna ligation system, by Promega (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Sanger sequencing, by Promega (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Top simple dyemix aliquot hot premix, by Enzynomics (1 mentions)
Top dna polymerase, by Bioneer (1 mentions)
Strataprep plasmid miniprep kit, by Agilent Technologies (1 mentions)
Pgem t easy cloning vector, by Promega (1 mentions)
Protoscript 2 first strand cdna synthesis kit, by New England Biolabs (1 mentions)
Abi 3500dx genetic analyzer, by Thermo Fisher Scientific (1 mentions)
10 protocols using expin gel sv
1
mtCOI DNA Amplification and Sequencing
2
Genomic DNA Extraction and PCR Amplification
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Genomic DNA was extracted from a human cell line HEK293A using DNeasy Blood & Tissue Kit (Qiagen), according to the manufacturer’s protocol. Genomic DNA was then used for PCR amplification using the primer list shown in Table 2 . The 20 µl PCR mix contained 10 µl of 2× TOPsimple DyeMix (aliquot)-HOT premix (Enzynomics, Korea), 7 µl of distilled water, 1 µl of gDNA template, and 1 µl of forward and reverse primers (10 pmole/µl) each. PCR conditions were as follows: initialization step at 95°C for 5 min, followed by 35 thermal cycles of 94°C for 40 s, annealing temperatures listed in Table 2 for 40 s, 72°C 40 s, and the final elongation step at 72°C 5 min. The PCR products were separated on a 1.5% agarose gel, and purified with ExpinTM Gel SV (GeneAll), according to the manufacturer’s protocol. The purified PCR products were cloned into a psi-CHECK2 vector (Promega, USA) using the LigaFastTM Rapid DNA Ligation System (Promega), as per the manufacturer’s protocol. The cloned plasmid DNA was isolated by using ExprepTM Plasmid SV, mini (GeneAll).
3
Sequencing and Locating UBC116 in Tomato
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The polymorphic DNA fragment of UBC116 was obtained from 'Youngmuja' (a resistant commercial cultivar) and sequenced as follows. The PCR band was excised from the agarose gel and purified using Expin TM Gel SV (GeneAll Biotechnology, Seoul, Korea); subsequently, the DNA fragment was directly sequenced using the dye-termination method of Genotech (Daejeon, Korea). A Basic Local Alignment Search Tool (BLAST) analysis of the DNA sequence was performed using the tomato reference genome (http://solgenomics.net/, ITAG2.3 release) to predict the physical location of UBC116.
4
Cloning and Purification of phlG Gene
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The phlG cloning primers (Supplementary Table 1 ) were designed with P. fluorescens Q2-87 reference sequences currently registered in NCBI [Locus_tag: PflQ2_2239]. The primers were synthesized by Cosmo Genetech (Seoul, Korea). The PCR mixture was 50 μl, comprising 2 mM of dNTPs, 2× PCR Buffer of KOD FX Neo, 1 μg of template DNA, 1.0 U KOD FX Neo, and 0.2 μM of each primer. The PCR reaction was performed as touchdown PCR, whose cycling began with a denaturation and annealing temperature of 83°C for 5 cycles, then reducing the annealing temperature 1°C every 5 cycles to 73°C, followed by 10 more cycles run at 73°C. Each primer was designed to amplify an 882-bp product, and the PCR product identified by gel electrophoresis in 1% agarose. Gel purification was done using Expin Gel SV (GeneAll, Seoul, Korea). To ligate with a donor vector, the purified PCR product underwent an A-tailing reaction (20 μl) as follows; dATP (NEB, Ipswich, MA, USA), 10× Reaction Buffer, and 5 units of TOP DNA Polymerase (Bioneer, Daejeon, Korea); the mixture was treated at 70°C for 30 min. DNA quantification was measured on a NanoDrop 2000C (Thermo Fisher Scientific, Waltham, MA, USA) to confirm its concentration and quality, after which it was stored at -20°C.
5
Genomic DNA Extraction and PCR Amplification
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The DNeasy Blood & Tissue Kit (Qiagen, Germany) was used to extract genomic DNA (gDNA) from human cell line HEK293A. gDNA was used for PCR amplification with 2 × TOP simple DyeMix (aliquot)-HOT premix (Enzynomics, Republic of Korea). The PCR mixture contained with 10 µL of contained with 2 × TOPsimple DyeMix, 7 µL of distilled water, 1 µL of gDNA template, and 1 µL of each primers (10 pmol/µL). The condition of PCR were as follow: an initialization at 95 °C for 5 min, 35 thermal cycles of 94 °C for 40 s, primer-specific annealing temperatures for 40 s, 72 °C for 40 s, and a final elongation step at 72 °C for 5 min. The products of PCR were separated on a 1.5% agarose gel and purified with Expin Gel SV (GeneAll, Republic of Korea). The purified PCR products were cloned into a psi-CHECK2 vector (Promega, USA) by LigaFast Rapid DNA Ligation System (Promega, USA). The Exprep Plasmid SV, mini (GeneAll, Republic of Korea) was used for plasmid isolation.
6
Phylotyping of B. glumae Isolates
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To analyze the phylotype of B. glumae isolates, the hrpB and tofI genes of 11 representative cluster isolates (RCIs) (R1, R5, R12, R13, R50, R65, R84, R93, R95, R96, and R121) taken from each Tnp-PCR cluster (I to XI) were amplified with primer pairs hrpB-1 (5′-ATGTTTTCGATGCTCTAT-3′) and hrpB-2 (5′-TCAGCGCTCGAGCGTCTC-3′) and tofI-NdeI (5′-GGCCATATGCAAACTTTCGTTCAC-3′) and tofI-XhoI (5′-GGCCTCGAGGGCCGCTTCGGGTTGCGA-3′), respectively (Kang et al. 2008; Kim et al. 2004 (link)). Then, PCR amplification was performed as described for the 16S rRNA gene region. The PCR products were purified using Expin Gel SV (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer's instructions.
Sequencing was performed at Macrogen (Daejeon, Korea) using the same primer pairs that were used for PCR amplification. Partial sequences were analyzed using the BLAST program of the National Center for Biotechnology Information reference database. A phylogenetic tree was constructed using the neighbor-joining method and Tajima-Nei distance model in MEGA X software (Kumar et al. 2018) (link).
Sequencing was performed at Macrogen (Daejeon, Korea) using the same primer pairs that were used for PCR amplification. Partial sequences were analyzed using the BLAST program of the National Center for Biotechnology Information reference database. A phylogenetic tree was constructed using the neighbor-joining method and Tajima-Nei distance model in MEGA X software (Kumar et al. 2018) (link).
7
Cloning and Sequencing of SmMKS2 cDNA
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The total RNA was isolated from the collected tissues and converted to cDNA using ProtoScript®II First Strand cDNA Synthesis kit (New England Biolabs, Massachusetts, USA) and oligo (dT)18 primer. Full-length cDNA sequences for SmMKS2s were obtained by polymerase chain reaction (PCR) using 1 μL of the resulting cDNA and gene-specific primers (Supplementary File 8 ). The PCR products were purified using Expin™ Gel SV (GeneAll Biotechnology Co., Ltd., Seoul, Korea) and cloned into pGEM-T Easy cloning vector (Promega, Wisconsin, USA). The recombinant plasmids were transformed into E. coli TOP10 competent cells. Positive clones were selected by blue/white colony screening and PCR colony. Recombinant plasmids were extracted using StrataPrep Plasmid Miniprep Kit (Agilent Technologies, California, USA) and their inserts were sequenced. Three SmMKS2 sequences have been deposited in GenBank with accession number MK990608, MK990609, and MK990610, respectively. Phylogenetic and molecular evolutionary analyses were conducted using MEGA X [22 (link)].
8
Generating V2 Deletion Plasmids
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To create the V2 deletion plasmids, the pGL4.11 vector cloned with the V2 original construct was amplified by PCR using 5′-phosphorylated primers; S, 5′-GGC ACA CAG AGA TAC GCG CA-3′, and AS, 5′-TGT GTG CGT GTA GGG GGT TA-3′ (Supplementary Fig. 4 ). PCR was performed with 12.5 µl of SmartGene 2 × pfu Mixed Taq Advanced (SJ Bioscience, Republic of Korea), 9.5 µl of nuclease-free water, 1 µl of primers (10 pmol/µl), and 1 µl of pGL4.11 vector cloned with V2 original construct as template DNA. The PCR conditions were as follows: initialization at 94 °C for 3 min, 22 thermal cycles of 94 °C for 40 s, primer-specific annealing at 61 °C for 30 s, 72 °C for 6 min, and a final elongation step at 72 °C for 5 min.
All PCR products were separated on a 1.5% agarose gel and purified with Expin Gel SV (GeneAll, Republic of Korea). The purified PCR products were cloned into a pGL4.11-T vector (Promega, USA) and ligated by LigaFast Rapid DNA Ligation System (Promega, USA). Plasmid isolation was performed with the Exprep Plasmid SV mini (GeneAll, Republic of Korea). The vector into which the PCR products were inserted was verified by colony PCR.
All PCR products were separated on a 1.5% agarose gel and purified with Expin Gel SV (GeneAll, Republic of Korea). The purified PCR products were cloned into a pGL4.11-T vector (Promega, USA) and ligated by LigaFast Rapid DNA Ligation System (Promega, USA). Plasmid isolation was performed with the Exprep Plasmid SV mini (GeneAll, Republic of Korea). The vector into which the PCR products were inserted was verified by colony PCR.
9
HCV Genotyping using Sanger Sequencing
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The PCR products of partial Core and NS5B were purified (ExpinGel SV, GeneAll Biotechnology, Seoul, Korea) and sequenced using an Applied Biosystems (ABI) PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit Version 3.1, the same PCR primers, and an ABI 3500 DX Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences were aligned with reference sequences retrieved from GenBank Database using ClustalX v.2.1 (Larkin et al. 2007 (link)). HCV genotype reference sequences were retrieved from the HCV database (http://hcv.lanl.gov/content/sequence/HCV/ToolsOutline.html ). The following sequences in GenBank were used as references in the phylogenetic analysis: M62321 and M67463(1a), D90208 and M58335(1b), D00944 and AB047639(2a), AB031663(2k), D17763, D28917(3a), D49374(3b), Y12083 and AY858526(6a), D84262(6b), D84263(6d), D84264(6k).
10
HCV Genotyping via Core Region Sequencing
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Genotype was determined based on the nucleotide sequence of the HCV core region. HCV RNA was extracted, and nested RT-PCR of the partial core region was performed on the samples with HCV RNA ≥ 12 IU/mL. Primer pairs of 954F/410 R were used in the first round, and 953F/951R were used in the second round, as previously described (Wasitthankasem et al., 2015 (link)). Target PCR amplicon of the core region was purified (ExpinGel SV; GeneAll Biotechnology, Seoul, Korea) and sequenced.
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