The size of the amplicon resulting from PCR amplification was verified prior to ensuring the correct size by electrophoresis on a 2% agarose gel and DNA fragments were visualized by gel red (Biotium, UK). PCR products were then purified to remove any unwanted salts as well as remaining primers and free nucleotides from the PCR products that might interfere with the outcome of sequencing results based on binding to the silica SV membrane using ExpinTM PCR SV Kit (GeneAll, Korea) in accordance with the manufacturer’s protocol. Briefly, 5 volumes of binding buffer (PB buffer), containing chaotropes, was added to the PCR product and mixed well. The sample mixture was transferred to the SV column and centrifuged (12,000 rpm for 30 s); then, 700 ul of washing buffer (NW buffer), containing ethanol, was added to the SV column and centrifuged again (12,000 rpm for 30 s). To complete removal of ethanol, the SV column was centrifuged for an additional 1 min and 50 μl of elution buffer (EB buffer, 10 mM Tris-HCl, pH=8.5) was added to the membrane center of the SV column and remained for 1 min at room temperature and then, centrifuged at 12,000 rpm for 1 min to elute the DNA. The eluted DNA was stored at −20°C until delivery for sequencing.
Expintm pcr sv kit
Manufactured by GeneAll
The ExpinTM PCR SV kit is a laboratory equipment product that facilitates the purification of PCR amplicons. It provides a streamlined process for the extraction and purification of DNA fragments from PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Nextera xt index kit protocol, by Illumina (3 mentions)
Miseq platform, by Illumina (3 mentions)
Accupower pcr premix kit, by Bioneer (3 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
Gelred, by Biotium (1 mentions)
Accupower hotstart pcr premix kit, by Bioneer (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
T7 rna polymerase, by New England Biolabs (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Hypervac vc2124, by Hanil (1 mentions)
7 protocols using expintm pcr sv kit
1
PCR Amplicon Purification and Sequencing
2
Fungal ITS2 Amplification and Sequencing
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The fungal internal transcribed spacer 2 (ITS2) region was amplified with primers ITS3 and ITS4 (White et al., 1990 (link)) with Illumina sequencing adaptors attached. PCR was conducted 3 times for each samples using AccuPower PCR PreMix kit (Bioneer, Daejeon, South Korea). PCR conditions were as follows: 94°C for 5 min, 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s, and 72°C for 10 min as final extension. PCR products were confirmed on 1% agarose gel (BIOFACT, Daejeon, South Korea) with gel electrophoresis. After purification using the ExpinTM PCR SV kit (GeneAll Biotechnology, Seoul, South Korea), a unique identifier sequence was attached to each PCR products with a second round PCR following the Nextera XT index kit protocol (Illumina, San Diego, CA, United States). Second PCR products were purified as above. Concentration of each amplicon library were measured using a NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, United States). Amplicon libraries were pooled in equimolar quantities and sequenced using Illumina MiSeq platform at Macrogen (Seoul, South Korea).
3
Fungal rRNA Amplification for MiSeq Sequencing
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For MiSeq sequencing, the 5.8S and ITS2 regions of the fungal rRNA gene were ampli ed with 5.8S-Fun and ITS4-Fun primers with adaptor sequences to avoid the ampli cation of plant DNA [44] . PCR ampli cation was performed in triplicate using an AccuPower PCR PreMix kit (Bioneer, Daejeon, South Korea). The rst round of PCR was conducted using to the following protocol: 96 °C for 5 min; 30 cycles of 94 °C for 30 s, 58 °C for 40 s, and 72 °C for 2 min; and 72 °C for 10 min for the nal extension. PCR products were separated on a 1% agarose gel and puri ed using an ExpinTM PCR SV kit (GeneAll Biotechnology, Seoul, South Korea). The second round of PCR was conducted using the same protocol, but cycles were reduced to 10, and the unique identi er sequences were attached following the Nextera XT index kit protocol (Illumina, San Diego, CA, USA). Products from the second PCR were checked and puri ed as described above. The concentration of each amplicon library was measured using NanoDrop2000 (Thermo Fisher Scienti c, Waltham, MA, USA) and then pooled in equimolar quantities.
Sequencing was performed on an Illumina MiSeq platform at Macrogen (Seoul, South Korea).
Sequencing was performed on an Illumina MiSeq platform at Macrogen (Seoul, South Korea).
4
Fungal rRNA Amplification for MiSeq Sequencing
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For MiSeq sequencing, the 5.8S and ITS2 regions of the fungal rRNA gene were ampli ed with 5.8S-Fun and ITS4-Fun primers with adaptor sequences to avoid the ampli cation of plant DNA [44] . PCR ampli cation was performed in triplicate using an AccuPower PCR PreMix kit (Bioneer, Daejeon, South Korea). The rst round of PCR was conducted using to the following protocol: 96 °C for 5 min; 30 cycles of 94 °C for 30 s, 58 °C for 40 s, and 72 °C for 2 min; and 72 °C for 10 min for the nal extension. PCR products were separated on a 1% agarose gel and puri ed using an ExpinTM PCR SV kit (GeneAll Biotechnology, Seoul, South Korea). The second round of PCR was conducted using the same protocol, but cycles were reduced to 10, and the unique identi er sequences were attached following the Nextera XT index kit protocol (Illumina, San Diego, CA, USA). Products from the second PCR were checked and puri ed as described above. The concentration of each amplicon library was measured using NanoDrop2000 (Thermo Fisher Scienti c, Waltham, MA, USA) and then pooled in equimolar quantities.
Sequencing was performed on an Illumina MiSeq platform at Macrogen (Seoul, South Korea).
Sequencing was performed on an Illumina MiSeq platform at Macrogen (Seoul, South Korea).
5
Fungal Species Identification from Egg Clutch
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To ensure correct species identification in 2017, a representative egg clutch with infection symptoms was sampled once and preserved in 70% ethanol until use. Before DNA extraction, the sample was rinsed twice with 100% ethanol to remove organic debris, and mycelia were carefully detached from the egg surface using a sterilized scalpel and tweezers. Genomic DNA of the mycelia was extracted using a modified cetyl trimethylammonium bromide method (Rogers & Bendich 1994) . The nuclear ribosomal internal transcribed spacer (ITS) region was amplified with the primer set ITS1oo (Riit et al. 2016 (link)) and ITS4 (White et al. 1990 (link)). PCR amplification was conducted using an AccuPower HotStart PCR PreMix kit (Bioneer) with 1 µl of template DNA and 1 µl of each primer. PCR denaturation was conducted at 95°C for 5 min, and amplification was done over 35 cycles at 95°C for 30 s, 55°C for 30 s and 72 °C for 40 s, followed by a final extension at 72°C for 10 min. The PCR products were checked on 1% agarose gel via electrophoresis and purified using an ExpinTM PCR SV kit (GeneAll Biotechnology). All sequencing was conducted at Cosmogenetech (Seoul, Korea).
The sequence was proofread and edited using MEGA5 (Tamura et al. 2011) (link). Initial identification was performed using BLAST against the NCBI data-
The sequence was proofread and edited using MEGA5 (Tamura et al. 2011) (link). Initial identification was performed using BLAST against the NCBI data-
6
SNP Identification via High-Fidelity PCR
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The genes with potential SNPs were amplified using Q5® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) according to manufacturer’s instructions. We used 20 ng/μL genomic DNA from SR8LDH and BK01 as template DNA. Amplified products were purified using Geneall ExpinTM PCR SV kit (Geneall, Seoul, Korea). Sanger sequencing was performed to confirm predicted SNPs (Cosmogenetech, Seoul, Korea).
7
Synthesis and Purification of Guide RNA for CRISPR
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Double-strand DNA (dsDNA) templates were synthesized with forward template DNA including T7 promoter and 20 bp crRNA sequence, reverse template DNA, Power-Pfu (500 U/µL, NanoHelix), and dNTPs (10 mM, Thermo Scientific). sgRNAs were synthesized by in vitro transcription using the dsDNA template, T7 RNA polymerase (50,000 units/mL, NEB), 50 mM MgCl2, 0.1 M DTT, rNTPs (100 mM, Jena Bioscience), and RNase inhibitor murine (40,000 units/mL, NEB) at 37 ℃ for 16 h, precipitated with isopropanol (Sigma) and purified with GeneAll ExpinTM PCR SV kit (GeneAll Biotechnology). sgRNA concentration was measured using NanoDrop 2000 (Thermo Scientific), and characterized by 1.0% denaturing formaldehyde gel electrophoresis with MOPS buffer (Biosolution). sgRNA was freeze-dried using HyperVAC VC2124 (Hanil Scientific) for long-term storage.
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