Genomic DNA was isolated from cultured cell lines using the G-DEX™ IIc Genomic DNA Extraction kit (iNtRON Biotechnology Inc., 17231, Seoul, Korea), following the manufacturer’s prescribed protocols. To detect the c.3140A > G (H1047R) missense mutation in the coding sequence of canine PIK3CA, the following sequencing primer sets, originated from a previous study [12 (link)], were used: F: 5’- CTG GAA TGC CAG AAC TAC AAT C -3′; R: 5’- CTG TTC ATG GAT TGT GCA ATT CC -3′. PCR products were electrophoresed, and specific bands were excised from the agarose gel. Amplified PCR products were extracted from the gel using an EZ-Pure™ Gel Extraction Kit. Ver. 2 (Enzynomics, Daejeon, Korea) DNA samples were submitted to Solgent Inc. (Daejeon, Korea) for sequencing using an ABI PRISM 3730XL Analyzer.
G dex iic genomic dna extraction kit
Manufactured by iNtRON Biotechnology
The G-DEX IIc genomic DNA extraction kit is a lab equipment product designed for the extraction and purification of genomic DNA from various biological samples. It utilizes a simple and efficient protocol to isolate high-quality genomic DNA.
Automatically generated - may contain errors
Lab products found in correlation
Abi 3730xl capillary dna sequencer, by Thermo Fisher Scientific (2 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (2 mentions)
Megaquick spin total fragment dna purification kit, by iNtRON Biotechnology (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Pgem t vector, by Promega (2 mentions)
Prism 3730xl analyzer, by Thermo Fisher Scientific (1 mentions)
Trueseq adaptors, by Illumina (1 mentions)
T7e1 endonuclease, by New England Biolabs (1 mentions)
Avidin tritc conjugate, by Merck Group (1 mentions)
7 protocols using g dex iic genomic dna extraction kit
1
Canine PIK3CA Mutation Detection by Sequencing
2
JAK-2 V617F Mutation Assay Protocol
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Genomic DNA was isolated with the G-DEX™IIc Genomic DNA Extraction Kit (iNtRON Biotechnology). JAK-2 mutation assay for V617F was assessed using a qualitative, real-time, PCR-based allelic discrimination assay, as described previously (16 (link), 17 (link)). PCR was performed on QuantStudio 5 Real-Time PCR analyzer (Applied Biosystems) in two separate tubes for normal and mutated alleles. In total, fifty nanograms of genomic DNA were amplified in a 40-cycle PCR at an annealing temperature of 62°C. All the reactions were performed in a final volume of 20 ul contacting SYBER Absolute ROX mix (Thermo Scientific©). PCR results were analyzed applying the delta Ct method. A cutoff of 1% or more of mutate alleles was chosen to define an individual as positive. The nominal estimated cost per test is estimated at 100 USD, which was fully financed by the public healthcare system.
3
Genomic DNA Extraction and CRISPR-Cas9 Analysis
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Genomic DNA was extracted from organ tissues by a G-DEX IIc genomic DNA extraction kit (Intron Biotechnology, Gyeonggi, Korea). Primers were designed for amplifying the LNP-CRISPR-mAT–targeted genomic region. Next, in T7E1 analysis, polymerase chain reaction (PCR) amplicons were subjected to hetero-duplex hybridization and 30 min of T7E1 endonuclease (New England Biolabs, Ipswich, MA, USA) incubation. The presence of the cut band in gel electrophoresis was designated as indel formation. For targeted deep sequencing, interesting genomic regions were amplified by PCR from genomic DNA extracted from transfected cells or LNP-injected tissues. The produced amplicons were barcoded during subsequent PCR with Illumina TrueSeq adaptors. The products were purified with a PCR purification kit (Geneall, Seoul, Korea) and then were pooled in an equimolar ratio. The final libraries were paired-end sequenced using Illumina Miseq v2 (PE150) (Illumina, San Diego, CA, USA). Indel frequencies were quantified using Cas-Analyzer (www.rgenome.net ). Indels in the region 3-bp upstream from the protospacer-adjacent motif sequence were considered mutations resulting from Cas9.
4
Radioactive Iodine DNA Extraction
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Each group of cells (5 × 106 cells per group) was incubated with 2.22 MBq of I-131 for 7 h, and DNA digested with proteinase K was extracted with a G-DEX IIc genomic DNA extraction kit (Intron Biotechnology, Seongnam, Republic of Korea) according to the manufacturer’s recommendations. Extracted DNA was run on a 1% agarose gel at 100 V for 30 min in TAE buffer.
5
DNA Oxidative Damage Quantification
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Cellular DNA was isolated using the G-DEX™ IIc Genomic DNA Extraction Kit (iNtRON Biotechnology, Inc., Sungnam, Kyungki-Do, Republic of Korea) and quantified by spectrophotometry. The amount of 8-hydroxy-2-deoxyguanosine (8-OHdG, the deoxyriboside form of 8-oxoG) in DNA was determined using the BIOXYTECH® 8-OHdG-EIA™ kit (OXIS Health Products, Inc., Portland, OR, USA) according to the manufacturer’s instructions. The amount of 8-oxoG was also estimated by a fluorescence-binding assay: cells were fixed and permeabilized with ice-cold methanol for 15 min and 8-oxoG was visualized with avidin-TRITC conjugate (Sigma-Aldrich) under a confocal microscope (Piao et al. 2011 (link)).
6
Bisulfite Sequencing of Genomic DNA
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Genomic DNA was isolated from cells using G-DEX IIc Genomic DNA Extraction Kit (iNtRON Biotechnology, Gyeonggi-do, Korea) according to the manufacturer’s protocol. Bisulfite conversion was performed using the EZ DNA Methylation—Gold Kit (ZYMO RESEARCH, Orange, CA, USA) according to the manufacturer’s protocol. Bisulfite-specific PCR reactions were carried out on a GeneAmp PCR System 9700 (Applied Biosystems) using the following protocol: 95°C for 15 minutes, 50 cycles of 95xC for 20 seconds, 55°C for 40 seconds, 72°C for 30 seconds, and extension at 72°C for 10 minutes. The primer sequences used for PCR are listed in S2 Table . PCR products were purified using the MEGAquick-spin Total Fragment DNA Purification Kit (iNtRON Biotechnology), cloned into pGEM T vector (Promega, Madison, WI, USA), and sequenced using an ABI 3730XL Capillary DNA sequencer (Applied Biosystems). Methylated or unmethylated states of CpG sites were determined from the sequence data by using QUMA (QUantification tool for Methylation Analysis) software [53 (link)].
7
Genome-Wide DNA Methylation Analysis
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Genomic DNA was isolated from cells using a G-DEX IIc Genomic DNA Extraction Kit (iNtRON Biotechnology, Gyeonggi-do, Korea) according to the manufacturer’s protocol. Bisulfite conversion was performed using an EZ DNA Methylation-Gold Kit (ZYMO RESEARCH, Orange, CA, USA) according to the manufacturer’s protocol. Bisulfite-specific PCR reactions were performed on a GeneAmp PCR System 9700 (Applied Biosystems) using the following protocol: 95 °C for 15 min, 50 cycles of 95 °C for 20 s, 55 °C for 40 s, 72 °C for 30 s, and extension at 72 °C for 10 min. The primer sequences used for PCR are listed in Supplementary Table 2 . PCR products were purified using a MEGAquick-spin Total Fragment DNA Purification Kit (iNtRON Biotechnology), cloned into pGEM T vector (Promega, Madison, WI, USA), and sequenced using an ABI 3730XL capillary DNA sequencer (Applied Biosystems). The methylation state of the CpG sites was determined from the sequence data by using QUMA (QUantification tool for Methylation Analysis) software60 (link).
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