After collection, DNA was isolated from the fresh caps using the DNeasy plant tissue kit (Qiagen). Isolations were carried out as described by the supplier. In brief, 100 mg of each mushroom cap was measured and homogenized with a pestle. A total of 400 μl AP1 buffer and 4 μl RNase A were added to a 1.5 ml Eppendorf tube. The samples were vortexed and incubated under rotation for 10 min at 65°C. After the incubation, 130 μl of P3 buffer was added and samples were incubated on ice for 5 min. Subsequently, samples were centrifuged for 5 min at 20,000g and supernatants were transferred to a Qiashredder spin column (Qiagen). Samples were then recentrifuged at 20,000g for 2 min. The flow‐through was transferred to a clean tube. A total of 1.5 volumes of buffer AW1 was added and 650 μl of this mixture was transferred to a DNeasy Mini spin column (Qiagen). Further isolation was exactly as described by the manufacturer.
Dneasy plant tissue kit
Manufactured by Qiagen
Sourced in United Kingdom
The DNeasy Plant Tissue Kit is a product designed for the isolation and purification of DNA from a variety of plant tissues. It utilizes a silica-based membrane technology to efficiently capture and purify DNA, ensuring high-quality DNA samples are obtained for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Qiashredder spin column, by Qiagen (1 mentions)
Dneasy mini spin column, by Qiagen (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Hiseq 2000 system, by Illumina (1 mentions)
Kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Taq polymerase, by Merck Group (1 mentions)
Nanodrop, by Eppendorf (1 mentions)
7 protocols using dneasy plant tissue kit
1
Mushroom DNA Extraction Protocol
2
Potato Genomics: Sequencing and Analysis
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Samples were obtained from the US Department of Agriculture potato gene bank and included South American wild species and landrace accessions germinated from seed and North American cultivars as in vitro clones (Dataset S1 ). Single individuals were selected from accessions to represent populations. DNA was purified from leaves using the Qiagen DNeasy Plant Tissue Kit. Illumina-compatible paired-end sequencing libraries (500-nt fragment size) were prepared and sequenced in paired-end mode (125 nt) to 8× genome coverage (diploids) and 16× coverage (tetraploids) on an Illumina HiSeq 2500 system at the Michigan State University Research Technology Support Facility Genomics Core; one library (Superior) was sequenced in paired-end mode to 150 nt. A subset of libraries was sequenced in paired-end mode (100 nt) on an Illumina HiSeq 2000 system (Dataset S1 ).
3
Comprehensive Pterocarya Species Phylogeny
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In this study, we used twenty-two individuals that represented all species and varieties and covered the entire range of each of the species (n: number of individuals, p: population number): P. fraxinifolia (n = 3, p = 3), P. hupehensis (n = 4, p = 4), P. macroptera var. delavayi (n = 2, p = 1), P. macroptera var. insignis (n = 1, p = 1), P. macroptera var. macroptera (n = 3, p = 3), P. rhoifolia (n = 3, p = 3), P. stenoptera (n = 3, p = 2), and P. tonkinensis (n = 3, p = 1) (Figure 6 ). Juglans mandshurica and Cyclocarya paliurus were used as outgroups. The voucher specimens are housed in the herbarium of the Shanghai Chenshan Botanical Garden (CSH), at Niigata University, and at Tarbiat Modares University (TMU). None of the field collections of Pterocarya species required specific permissions or involved endangered or threatened species.
DNA extraction was performed with a Qiagen DNeasy Plant Tissue Kit from silica-gel dried leaves according to the manufacturer’s (Qiagen, Valencia, CA, USA) standard protocol. The DNA extraction quality was checked by 1% agarose gel in conjunction with 1 KB Plus DNA Ladder (Invitrogen) or a New England Biolabs 100 bp DNA ladder marker (Ipswich, MA, USA). The genomic DNA concentrations were subsequently quantified with a dsDNA HS kit on a Qubit 2.0 Fluorometer.
DNA extraction was performed with a Qiagen DNeasy Plant Tissue Kit from silica-gel dried leaves according to the manufacturer’s (Qiagen, Valencia, CA, USA) standard protocol. The DNA extraction quality was checked by 1% agarose gel in conjunction with 1 KB Plus DNA Ladder (Invitrogen) or a New England Biolabs 100 bp DNA ladder marker (Ipswich, MA, USA). The genomic DNA concentrations were subsequently quantified with a dsDNA HS kit on a Qubit 2.0 Fluorometer.
4
Phylogenetic Analysis of Emmenopterys henryi
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We obtained silica-dried leaf material from 38 populations of Emmenopterys henryi throughout its range (Table S1 , Fig. 1A ; see Supplementary Method S1 for more details of the study species). In each population, representative samples of 10–20 plants were taken, resulting in a total of 433 individuals. Total genomic DNA was extracted from the dried leaf tissue using a DNeasy plant tissue kit (Qiagen). All samples were sequenced at three intergenic spacer (IGS) regions of chloroplast DNA (cpDNA), while a subset of individuals (n = 212) was also sequenced at the entire internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). Of these 433 individuals, 394 (representing 37 populations; population S23 with 1 individual was excluded) were surveyed for AFLPs (Table S1 ; Fig. 1A ). Pinckneya bracteata (Bartram) Raf., collected from the JC Raulston Arboretum (Raleigh, NC, USA), was selected as an outgroup for the phylogenetic analyses based on a previous molecular phylogenetic study of Rubiaceae33 . Voucher specimens of this species and all sampled populations of E. henryi are stored at the Herbarium of Zhejiang University (HZU; Hangzhou, Zhejiang, China).
5
Apple Rootstock Genome Sequencing and Assembly
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High quality DNA was extracted from the apple rootstocks ‘M.9’, ‘M.27’, and ‘M.116’ using the Qiagen DNeasy plant tissue kit following the standard protocol (Qiagen, UK). The Genome Analysis Centre (TGAC), UK, performed DNA library preparation for 100bp paired end (PE) read sequencing using standard Illumina chemistry and sequenced on an Illumina HiSeq 2000 generating a minimum of ×50 coverage. The average insert size of the libraries was 621bp. Low quality reads were removed using fastq-mcf (Aronesty, 2013 ). The sequence reads were aligned to the reference apple genome: ‘Golden Delicious’ Malus × domestica v1.0 pseudo haplotype downloaded from the Genome Database for Rosaceae (GDR) (Jung et al., 2008 (link); Velasco et al., 2010 (link)) using reference-guided assembly (RGA). The commercial software Geneious® was used for RGA, multiple chromosome alignment and data visualization (www.geneious.com ).
6
Dermatophyte Genomic DNA Extraction and Multiplex PCR
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The genomic DNA of the dermatophytes was extracted using the Qiagen DNeasy Plant Tissue Kit (QIAGEN, Germany). Following quality confirmation by Nanodrop (Eppendorf, Germany), specific primers for target genes were designed by the Gene runner software and blasted on the NCBI website to confirm specificity. Multiplex PCR was used to detect isolates harboring Mep1-5, Erg11, 24, 26, ScpA, B, and NPII genes. The reaction mixture of PCR amplification was adjusted to 50 μl, which included 50 ng of genomic DNA solution, 10× PCR buffer, 0.6 U of Taq polymerase (Merck, Germany), 0.1 mM of dNTPs, and 0.5 mM of the primer (Table 1 ). The temperature steps of the PCR reaction were performed as follows: initial denaturation step at 95°C for 5 min in 35 cycles, including 95°C denaturation for 30 sec, and primer binding at 56°C for 30 sec. The amplification step was performed at 72°C for 1 min; after 35 cycles, the final amplification step was carried out at 72°C for 10 min. PCR products were electrophoresed on 1.5% agarose gel in the presence of positive and negative controls, stained with erythrogel, and photographed by a gel documentation device.
7
Itraconazole-resistant Aspergillus Fumigati cyp51A Sequencing
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All of the itraconazole-resistant Aspergillus section Fumigati isolates were subjected to cyp51A gene sequencing. Genomic DNA was extracted by using Dneasy plant tissue kit (Qiagen, Crawley, UK). The cyp51A coding region was amplified by PCR using the primer set P450-A1 (5’-ATGGTGCCGATGCTATGG-3) and Cyp51AR2 (5’-AGTGA ATAGAGGAGTGA ATCC-3’) and sequenced as described in Prigitano et al. [19 (link)]. The cyp51A gene promoter was amplified and sequenced using the primers P-A7 and P-A5 as previously described in Mellado et al. [27 (link)]. Sequence alignment was performed using the ClustalW algorithm (www.ebi.aci.uk ). The cyp51A sequence from A. fumigatus strain 237 (GenBank accession number: AF338659) was used as a wild type reference.
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