The green and white leaves of R. japonica, J. chinensis, and C. pisifera were individually ground in liquid nitrogen using a mortar and pestle. Each region from one leaf was excised with a razor blade and ground independently for plants with green and white sectors within the same leaf (Fig. 1a ). Total genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) protocol [53 (link)] or a DNA extraction kit (Exgene Plant SV mini kit, GeneAll Biotechnology, Seoul, Korea) following the manufacturer’s instructions. The quality and concentration of the extracted DNA were determined with a UV spectrophotometer (Nanodrop ND-1000; Thermo Fisher Scientific, Waltham, MA, USA) and by agarose gel electrophoresis. Paired-end DNA sequencing libraries were generated using a TruSeq Nano kit (Illumina, San Diego, CA, USA) and subsequently sequenced by Lab Genomics Inc. (Seongnam, Republic of Korea) using an Illumina MiSeq genome instrument, according to the standard protocol provided by the manufacturer. The sequences and plastid information for the albino leaf tissue (ALT) of E. hamiltonianus (Number 17 in Fig. 1 ) were identical to that of normal tissue from our previous report [30 ].
Exgene plant sv mini kit
Manufactured by GeneAll
The Exgene Plant SV mini kit is a laboratory equipment product designed for the rapid and efficient extraction of high-quality genomic DNA from plant samples. The kit utilizes a simple and reliable silica-membrane-based method to isolate DNA, making it suitable for a wide range of plant species.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Truseq nano kit, by Illumina (1 mentions)
Gotaq dna polymerase, by Promega (1 mentions)
Dntp mix, by Bioneer (1 mentions)
Exgene plant sv midi kit, by GeneAll (1 mentions)
Nanodrop 2000 spectrometer, by Thermo Fisher Scientific (1 mentions)
Miseq platform, by Illumina (1 mentions)
Taq polymerase, by GeneAll (1 mentions)
6 protocols using exgene plant sv mini kit
1
Genomic DNA Extraction and Sequencing
2
Molecular Characterization of Wheat Cultivars
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Genomic DNA was extracted from 50 mg of wheat flour for each wheat cultivar using the GeneAll Exgene Plant SV mini kit (GeneAll, Seoul, Korea) following the manufacturer’s instructions. The extracted genomic DNA was quantified and its quality assessed on a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and then it was diluted to 50 ng/μL. PCR analysis to discriminate between 1Bx7 and 1Bx7OE was performed using the method described by [5 (link),17 (link)]. PCR was performed in a reaction volume of 20 μL using 150 ng of genomic DNA, 1.25 U of Go Taq DNA polymerase (Promega, USA), 1× Green Go Taq reaction buffer (containing 1.5 mM MgCl2), 200 μM of dNTP mix (Bioneer, Daejeon, Korea) and 10 pmol each of forward and reverse primers. Left junction primers were: forward 5′-ACGTGTCCAAGCTTTGGTTC-3′ and reverse 5′-GATTGGTGGGTGGATACAGG-3′, and right junction primers were forward 5′-CCACTTCCAAGGTGGGACTA-3′ and reverse 5′-TGCCAACACAAAAGAAGCTG-3′ [17 (link)]. Amplification conditions for PCR were an initial cycle at 95 °C for 5 min, followed by 34 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 1 min, and then a final extension at 72 °C for 5.25 min. PCR products were resolved on 1.5–2.0% agarose gels in 0.5× Tris borate EDTA (TBE) buffer, stained with ethidium bromide and visualized under UV.
3
Transcriptome Sequencing of Euonymus Species
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Thirty-one E. hamiltonianus, one E. europaeus, and one E. japonicus were collected from various sources including wild and commercial samples (S6 Table ). The collected samples from Hantaek Botanical Garden (Baegam, Cheoin, Yongin, Gyeonggi, Korea) and Sannae Botanical Garden (Byeongcheon, Dongnam, Cheonan, South Chungcheon, Korea) were collected with permission from garden authorities (Permitted by Taek Joo Lee and Myung Hyoe Kim, president of Hantaek Botanical Garden and Sannae Botanical Garden, respectively). For samples collected from the wild, no specific permission was required for collecting the species in this study, according to the national and local legislations. Leaf samples were collected, flash frozen, and used for genomic DNA extraction using an Exgene Plant SV Midi Kit and an Exgene Plant SV Mini Kit (Geneall Biotechnology, Seoul) following the manufacturer’s protocols. The concentration and quality of the extracted genomic DNA were examined by gel electrophoresis and with a Nanodrop 2000 spectrometer (Thermo Scientific, USA). Among all 33 accessions, three E. hamiltonianus, one E. europaeus and one E. japonicus were used to generate paired-end (PE) libraries. The libraries were sequenced using the Illumina MiSeq platform at Phyzen (www.phyzen.com , Seongnam, Republic of Korea). This analysis yielded 0.82–1.20 Gbp of sequencing data per sample.
4
Genotyping of Watermelon Inbreds
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Seeds of a total of 21 watermelon inbreds including 10 CY and 11 red-fleshed lines R were achieved from the Plant Genetics and Breeding Research Center at Pusan National University (Miryang, South Korea) and Partner Seed Company. Genomic DNA was extracted from the first leaf of the seedling using the GeneAll Exgene Plant SV mini kit (GeneAll, Seoul, Korea). Genotyping was performed by using two LCYB gene (C locus)-based markers (Lcyb and Clcyb.600) (Bang et al., 2007 (link)) and a C2 locus-linked marker M7 developed in this study.
5
Genomic DNA Extraction from CRMs
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Genomic DNA was extracted from the CRMs and dried tissue samples collected under the monitoring project using the Exgene Plant SV mini kit (GeneAll, Korea), according to the manufacturer's instruction. The concentration of DNA isolated from each sample was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The final concentration of genomic DNA was adjusted to 100 ng/µL for analysis using the newly developed multiplex PCR; the DNA samples were stored at -20 • C until use.
6
Hibiscus DNA Isolation and Amplification
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Leaves of the five different Hibiscus cultivars were placed in liquid nitrogen for 1 min and crushed into powder. Genomic DNA was isolated using the Exgene ™ Plant SV mini kit (GeneAll Biotechnology Co., Seoul, Korea), according to the manufacturer's instructions. The primer pairs used for the amplification of trnL-F region were synthesized by Macrogen (Seoul, Korea); the sequences of the universal primers are listed in Table 2 [16] (link). The PCR reaction mixture consisted of 50 ng of DNA, 0.5 µM of primers, 10 µL of Taq polymerase (GeneAll Biotechnology Co., Seoul, Korea), and 5 µL of distilled water. PCR products were analyzed by electrophoretic separation in a submarine horizontal agarose slab gel apparatus. For running the DNA samples, a gel strength of 2% agarose was dissolved in 0.5 X TAE buffer followed by heating in microwave oven. After cooling to approximately 55 °C, 0.05 µL/mL GelGreen ® Nucleic Acid Gel Stain (Fremont, CA, USA) was added. Then, 300 mL of 0.5X TAE buffer was poured to sufficiently submerge the gel in the electrophoresis tank. Electrophoresis was performed at a constant voltage of 5 V/cm until the bromophenol dye reached almost the end of the gel. A UV-trans illuminator was used to visualize the DNA fragments of different sizes separated in the gel (Fig. 1).
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