The forward and reverse primers for the amplification of the KRAS mutations (G12D and G13D) were synthesized at the usual length of approximately 24 bps (Supplementary Table 1 ). The end-point PCR process comprised an initial denaturation step at 95°C for 15 min, followed by 35 cycles at 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s, as well as a final elongation step at 72°C for 7 min. Moreover, 5 μL of DNA was amplified in a total volume of 25 μL, containing 10X PCR buffer (Qiagen), 2.5 mM MgCl2, 0.25 mM deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of Taq DNA polymerase (Qiagen). Gel electrophoresis was used to separate the PCR products on a 2% agarose gel containing ethidium bromide (EtBr). The gel was visualized using a GelDoc System (Clinx Science Instruments). For direct sequencing of DNA, all the DNA samples were amplified (annealing temperature of 55°C) with the sequencing primer of KRAS exon 2, and were then purified by using the Expin PCR SV (GeneAll, Korea). The purified samples were directly sequenced using BigDyeTerminal chemistry, with the forward sequencing primer. The DNA sequencing reaction mixtures were electrophoresed using ABI's 3730XL DNA analyzer (Applied Biosystems, USA) at the Macrogen sequencing center (Macrogen Inc. Seoul, Korea).
Expin pcr sv
Manufactured by GeneAll
The Expin PCR SV is a laboratory instrument used for the amplification of DNA or RNA sequences through polymerase chain reaction (PCR) technology. It is a specialized device designed for efficient and reliable DNA/RNA amplification in a variety of applications.
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Lab products found in correlation
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (4 mentions)
Taq dna polymerase, by Qiagen (2 mentions)
Pcr buffer, by Qiagen (2 mentions)
Abi bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Ex taq dna polymerase, by Takara Bio (2 mentions)
Miniseq, by Illumina (2 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Rna clean and concentrator 5 kit, by Zymo Research (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Nextseq 500 platform, by Illumina (2 mentions)
Nextseq 500 550 mid output reagent cartridge v2 300 cycle, by Illumina (2 mentions)
Qubit dna hs assay kit, by Thermo Fisher Scientific (2 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Geldoc system, by Clinx (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Qubit 3 fluorometer, by Thermo Fisher Scientific (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Truseq ht dual index primers, by Illumina (1 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Gelred, by Biotium (1 mentions)
18 protocols using expin pcr sv
1
Amplification and Sequencing of KRAS Mutations
2
Direct Sequencing of C. burnetii DNA
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For direct sequencing of DNA, all DNA samples were amplified with primers for C. burnetii, and then purified by using Expin PCR SV (GeneAll, Korea). The purified samples were directly sequenced using BigDyeTerminal chemistry with the forward primer of Q-fever_IS111. We used Macrogen sequencing service (Macrogen Inc. Korea), through which the DNA sequencing reactions were electrophoresed on ABI's3730XL DNA Analyzers (Applied Biosystems, USA), which produces read lengths of 800–1000 bases.
3
DNA Template Preparation for KRAS Mutation
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To confirm the LOD and efficiency for HSSP, mutation DNA templates were made using DNA extracted from the AGS and HCT116 cell lines. The DNA was extracted using the QIAamp DNA Mini Kit (Qiagen). In addition, a template was made with DNA obtained from a sample of a patient diagnosed with the wild-type. The DNA template has a length of 777 bp containing KRAS mutation. DNA templates were made by PCR analysis using a designed primer and were then purified using the Expin PCR SV (GeneAll). The concentration and quality of the produced DNA templates were assessed using a Qubit 3 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and the DNA copy number was calculated using the EndMemo DNA/RNA copy number calculator. The DNA template was stored at –20 °C until use.
4
SNP Validation by Sanger Sequencing
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Common SNPs were selected by comparing the nucleotide sequences of the SNP loci of the samples of the same variety, and the polymorphic SNP loci were selected by comparing common SNPs among the varieties. When selecting common SNPs in the same variety, ≤50% missing data were allowed.
A subset of selected polymorphic SNPs was used for validation by Sanger sequencing. Primers were designed to produce 500–600 bp amplicons containing at least one putative SNP using Primer3 v. 2.3.5 [28 (link)]. Each 20 μL PCR reaction comprised 1× PCR buffer, 2.5 U Ex-Taq DNA polymerase (TaKaRa), 250 μM each dNTP, 10 pmol each SNP marker, and 20 ng genomic DNA template. PCR was performed using an ABI 2720 Thermocycler as follows: 10 min of denaturation at 95°C, 35 cycles (45 s at 94°C, 45 s at 58°C, and 45 s at 72°C) of amplification, and a final extension for 5 min at 72°C. The PCR products were assessed by 2% agarose gel electrophoresis and purified with Expin PCR SV (GeneAll Biotechnology, Seoul, ROK). The purified PCR products were amplified using an ABI BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems), and run on an ABI 3730XL DNA analyzer (Applied Biosystems). The acquired bidirectional sequences and the reference sequence were aligned in SeqMan software (DNASTAR).
A subset of selected polymorphic SNPs was used for validation by Sanger sequencing. Primers were designed to produce 500–600 bp amplicons containing at least one putative SNP using Primer3 v. 2.3.5 [28 (link)]. Each 20 μL PCR reaction comprised 1× PCR buffer, 2.5 U Ex-Taq DNA polymerase (TaKaRa), 250 μM each dNTP, 10 pmol each SNP marker, and 20 ng genomic DNA template. PCR was performed using an ABI 2720 Thermocycler as follows: 10 min of denaturation at 95°C, 35 cycles (45 s at 94°C, 45 s at 58°C, and 45 s at 72°C) of amplification, and a final extension for 5 min at 72°C. The PCR products were assessed by 2% agarose gel electrophoresis and purified with Expin PCR SV (GeneAll Biotechnology, Seoul, ROK). The purified PCR products were amplified using an ABI BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems), and run on an ABI 3730XL DNA analyzer (Applied Biosystems). The acquired bidirectional sequences and the reference sequence were aligned in SeqMan software (DNASTAR).
5
Comparative Analysis of 16S rRNA Sequences in Actinidia chinensis
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Partial sequences (570 bp) of the 16S rRNA gene were analyzed from A. chinensis individuals from Korea (n = 96) and China (n = 96). The target DNA was amplified using the 16S rRNA 16Sbr universal primer (5′-CCGGTCTGAACTCAGATCACGT-3′) and 16Sar universal primer (5′-CGCCTGTTTATCAAAAACAT-3′) set whose melting temperatures (Tm) are 55.7°C and 53.9°C, respectively. Polymerase chain reaction (PCR) was performed using a thermocycler (PTC-2040; Bio-Rad, Hercules, CA, USA) in 20-μL volumes with 10 to 20 ng of DNA, 0.5 units of DNA polymerase (Anti-HS Taq, TNT Research, Seoul, Korea), 250 μM of each dNTP, and 1× PCR buffer containing 2 mM MgCl2 and 10 pmol of each primer. The PCR amplification consisted of an initial denaturation at 95°C for 10 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The PCR fragments were purified using Expin PCR SV (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. Approximately 8 to 20 ng of purified product was used as a template for sequencing using the ABI BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster, CA, USA).
6
Targeted Deep Sequencing for Genome Editing
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On-target sites were amplified from genomic DNA using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific Korea) for targeted deep sequencing. The region of interest was amplified using the primer pairs listed in Table S9 . Illumina TruSeq HT dual index primers were used to label each PCR amplicon. The PCR products were purified using Expin PCR SV (Geneall Biotechnology, Korea). Pooled libraries were sequenced using MiniSeq (Illumina, San Diego, CA). Substitutions and indel frequencies were calculated by MAUND, which is available at https://github.com/ibs-cge/maund . Wild-type and mutant sequences were discriminated based on the presence of the single nucleotide missense mutation in the allele. Sequences carrying indels were counted as genome edited sequences. Indel-harboring sequences that could not be discriminated as wild-type or mutant sequences were counted as mutant sequences, because the percentage of wild-type-specific indels among all alleles was low. A-to-G conversion ratios in the mutant sequences were determined to calculate base editing frequency.
7
Direct Sequencing of C. burnetii DNA
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For direct sequencing of DNA, all samples were amplified with primers specific for C burnetii and then purified using Expin PCR SV (GeneAll, Seoul, Korea). Purified samples were sequenced directly using BigDye Terminator chemistry and forward primer Q-fever_IS111. Sequencing was performed by Macrogen sequencing service (Macrogen Inc, Seoul, Korea), which examined the DNA sequencing reactions on an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA), which produces read lengths of 800 to 1000 bases.
8
DENV-2 Whole Genome Sequencing Protocol
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All virus strains isolated from patient sera were subjected to whole genome sequencing. RNA was reverse transcribed using the Superscript III First-strand Synthesis System (Life Technologies, Carlsbad, USA). Each DENV-2 whole genome was amplified in 6 fragments, together with 5’ and 3’ untranslated regions (UTRs) using the Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific Inc., Pittsburgh, USA) as described elsewhere [25 (link)]. Amplified products were visualized in 2% agarose gels stained with GelRed (Biotium Inc., CA, USA) and were subsequently purified by using Expin PCR SV (GeneAll Biotechnology, Seoul, Korea). Sequencing was performed at a commercial facility according to the BigDye Terminator Cycle Sequencing kit (Life Technologies, Carlsbad, USA) protocol. Sequences were deposited in Genbank database under the accession numbers from KM279513 to KM279610.
9
Fungal ITS Sequence Amplification and Analysis
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The ITS1-5.8S-ITS2 region was PCR amplified from the isolates’ genomic DNA in a 25 µl reaction consisting of 10× PCR buffer, 10 µM each of ITS1 (5′-TCCGTAGGTGAACCT GCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers (White et al., 1990 ), 25 mM MgC12, 2 mM deoxynucleoside triphosphate, 2.5 unit of HotStarTaq DNA polymerase, and 10 µg of each genomic DNA. The PCR was performed for 30 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 60 s. The PCR products were then purified using Expin™ PCR SV (GeneAll, Korea), and confirmed by Sanger sequencing (First Base Laboratories Kuala Lumpur, Malaysia). TraceTuner version 3.0.6 (Denisov, Arehart & Curtin, 2004 ) was used for base and quality calling of the sequenced ITS. The low-quality called bases (Phred value < 20) of both ends of the sequences were then trimmed by running Lucy version 1.20 (Chou & Holmes, 2001 (link)) and the included zapping.awk script. The processed ITS sequences were searched against the NCBI non-redundant (nr) nucleotide database using the nucleotide BLAST program.
10
ATAC-seq protocol for chromatin accessibility
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ATAC-seq was performed as previously described57 (link). Briefly, cells were lysed in NLB buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.05% NP-40 and protease inhibitors (Sigma, P2714). Transposition reaction was performed on 105 nuclei using 5 μl of Nextera TDEI enzyme (Illumina, FC-121-1030) for 30 min at 37 °C. DNA was then purified by Expin PCR SV (GeneAll, 103-102) and the library was amplified using NEBNext High-Fidelity 2× PCR Master Mix (New England Biolabs, M0541). The libraries were size-selected by a gel-free double-sided size-selection using Agencourt AMPure XP beads (Beckman, 63881), at 0.5X and 1.2X. Library concentration was measured by DNA High Sensitivity Kit (Invitrogen) on a Qubit fluorometer (Invitrogen). Library quality and fragment sizes were assessed on a Bioanalyzer (Agilent). ATAC-Seq libraries from two biological replicas for each condition were sequenced on Illumina Hi-seq 2000 platform.
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