Fresh leaf samples of six Amana species, five from China and one from Japan (Table 1 ), were field-collected and dried with silica gel. Voucher herbarium specimens were deposited at the Herbarium of Zhejiang University (HZU). We extracted total DNA from ca. 3 mg of the silica-gel dried leaf tissue for each species using DNA Plantzol Reagent (Invitrogen) and following the manufacturer's protocol. The qualities and quantities of genomic DNA were checked on an Agilent BioAnalyzer 2100 (Agilent Technologies). Short-insert (500 bp) paired-end libraries were generated by using Genomic DNA Sample Prep Kit (Illumina) according to the manufacturer's protocol. Genomic DNA of each species was indexed by barcode tags and then pooled together for sequencing in one lane of HiSeq™ 2500 (Illumina, San Diego, California, USA) at Beijing Genomics Institute (BGI, Shenzhen, China).
Genomic dna sample prep kit
Manufactured by Illumina
Sourced in United States, China
The Genomic DNA Sample Prep Kit is a laboratory instrument designed to extract and purify genomic DNA from a variety of sample types. The kit includes reagents and consumables necessary for the DNA extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (4 mentions)
Hiseq 2500, by Illumina (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nextera mate pair sample preparation kit, by Illumina (2 mentions)
Dneasy plant mini kit, by Qiagen (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Genome analyzer iix, by Illumina (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rna fragmentation kit, by Thermo Fisher Scientific (2 mentions)
Dna plantzol reagent, by Thermo Fisher Scientific (1 mentions)
Qiaquick spin column, by Qiagen (1 mentions)
Klenow dna polymerase, by Illumina (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Minelute column, by Qiagen (1 mentions)
Hiseq 2000 sequencer, by Illumina (1 mentions)
Genome analyzer 2 ga 2, by Illumina (1 mentions)
Te buffer, by Nippon Gene (1 mentions)
24 protocols using genomic dna sample prep kit
1
Amana Species Genomic Sequencing
2
Illumina Whole-Genome Chromatin IP Sequencing
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The purified DNA products were modified for Illumina Whole-Genome Chromatin IP sequencing using an Illumina Genomic DNA Sample Prep kit as follows: size-selected DNAs were end-repaired by T4 DNA polymerase and phosphorylated by T4 DNA polymerase and T4 polynucleotide kinase. The products were incubated with Klenow DNA Polymerase (Illumina) to generate 3′ adenine overhangs and then ligated to Illumina adapters, which contain 5′ thymine overhangs. The adapter-ligated products were purified on QIAquick spin columns (Qiagen), PCR-amplified with Phusion DNA Polymerase (Finnzymes) for 30 cycles using an Illumina genomic DNA primer set. The PCR products were purified on QIAquick and MinElute columns (Qiagen).The quality of the DNA was assessed and quantified using an Agilent DNA 1000 Series II assay and NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and the DNA was diluted to 10 nM. Cluster generation and sequencing were performed using a Standard Cluster Generation kit and a Cycle Solexa Sequencing kit on the Illumina Cluster Station and Illumina HiSeq2000 sequencer following the manufacturer's instructions. Sequencing was carried out by the Research & Cooperation Division, BGI-Shenzhen.
3
Bacterial Genomic DNA Extraction and Sequencing
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Mixtures of bacterial genomic DNA were extracted using SepaGene (Sanko Junyaku, Tokyo, Japan) according to the manufacturer’s instructions. After samples were treated with 100 µg ml−1 ribonuclease (DNase-free) solution (Nippon Gene, Tokyo, Japan) in TE buffer (Nippon Gene, pH 8.0) at 37°C for 30 min and stored at 4°C for 3 days, the genomic DNA was extracted using phenol/chloroform method and recovered by ethanol precipitation.
A DNA library of approximately 600-bp was prepared using a genomic DNA sample prep kit (Illumina, San Diego, CA, USA), and DNA clusters were generated on a slide using a cluster generation kit (ver. 2) in the Illumina cluster station according to the manufacturer’s instructions. To obtain approximately 1×107 clusters per lane, the general procedure as described in the standard recipe was employed in the following order: template hybridization, isothermal amplification, linearization, blocking, denaturation, and hybridization of the sequencing primer (Illumina). All sequencing runs for 80-mers were performed with an Illumina Genome Analyzer II (GA II) using the Illumina sequencing kit (ver. 3). Fluorescent images were analyzed using the Illumina base-calling pipeline 1.5.0 to obtain FASTQ-formatted sequence data.
A DNA library of approximately 600-bp was prepared using a genomic DNA sample prep kit (Illumina, San Diego, CA, USA), and DNA clusters were generated on a slide using a cluster generation kit (ver. 2) in the Illumina cluster station according to the manufacturer’s instructions. To obtain approximately 1×107 clusters per lane, the general procedure as described in the standard recipe was employed in the following order: template hybridization, isothermal amplification, linearization, blocking, denaturation, and hybridization of the sequencing primer (Illumina). All sequencing runs for 80-mers were performed with an Illumina Genome Analyzer II (GA II) using the Illumina sequencing kit (ver. 3). Fluorescent images were analyzed using the Illumina base-calling pipeline 1.5.0 to obtain FASTQ-formatted sequence data.
4
Plastome Sequencing of Poikilospermum stipuleanatus
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Total genomic DNA was isolated from the silica-gel-dried leaves of P. stipuleanatus, collected in Maguan County, Yunnan Province, China, using a modified CTAB method (Doyle and Doyle, 1987 , Yang et al., 2014 (link)). Voucher specimens (JYH-2016,466) were deposited in the Herbarium of the Kunming Institute of Botany, CAS (KUN). Genomic DNA was randomly fragmented into 400–600 bp with an ultrasonicator. Short-insert (500 bp) paired-end libraries were constructed using the Genomic DNA Sample Prep Kit (Illumina), according to the manufacturer's protocol, and then sequenced on the Illumina HiSeq 2500 system at BGI (Shenzhen, Guangdong, China). The Illumina raw data were filtered with a NGS QC Toolkit (Patel and Jain, 2012 ). High-quality reads were assembled into contigs using SPAdes v3.10.1 (Nurk et al., 2013 ) with its default parameters. The representative plastome sequence contigs were then mapped onto the reference plastome sequence of P. japonicus (Genbank accession number: KX247146 ) in Bowtie v2.2.6 (Langmead and Salzberg, 2012 (link)) with its default-preset options. Assemblies were then assessed and connected using Bandage (Wick et al., 2015 (link)). The validated complete plastome sequences were deposited in GenBank (Table S1 ).
5
Genome Sequencing of Sibling Inbred Line
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A sibling inbred line, Sp75, was grown under standard greenhouse conditions with a 16-h light (27 °C) and 8-h dark (19 °C) cycle. Young leaves from 20-days-old plants were collected, immediately frozen in liquid nitrogen and stored at −80 °C. DNA was extracted using the QIAGEN DNeasy Plant Mini Kit following the manufacturer's instructions (QIAGEN, Valencia, CA, USA). DNA quality was evaluate via agarose gel electrophoresis and its quantity was determined on a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). Paired-end libraries with insert sizes of 150 bp, 200 bp, 300 bp, 500 bp and 1 kb and mate-pair libraries with insert sizes of 3, 10 and 15 kb were constructed using the Genomic DNA Sample Prep kit and the Nextera Mate Pair Sample Preparation kit (Illumina, San Diego, CA, USA), respectively, following the manufacturer's instructions, and sequenced on an Illumina HiSeq 2500 platform with paired-end mode.
6
Extraction and Sequencing of Entamoeba Genomic DNA
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Genomic DNA, from strains KU27 and KU50, was extracted using a QIAamp DNA Mini Kit (Qiagen, Hidden, Germany), according to the manufacturer’s instructions. Trophozoites of KU27 and KU50 were cultured in 25 cm2 tissue culture flasks (2–6 flasks) to obtain 4 to 9 μg of genomic DNA. Approximately 4–5×106 amebae were obtained per flask. Purified genomic DNA samples were subjected to next-generation sequencing using an Illumina Genome Analyzer IIx DNA Sequencer (GAIIx; Illumina, San Diego, CA). Genomic DNA libraries were constructed using a genomic DNA Sample Prep Kit (Illumina). DNA clusters were generated on a slide, using a Cluster Generation Kit v4 on an Illumina cluster station, according to the manufacturer’s instructions. All sequencing runs, for 125-mers, were performed using an Illumina GAIIx and Illumina Sequencing Kit (v5).
7
Genome Sequencing and Annotation of Chlamydia-like Bacteria
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Bacterial 1 kb insert DNA libraries (Purified Neochlamydia S13 and Protochlamydia R18) were prepared using a genomic DNA Sample Prep Kit (Illumina, San Diego, CA). DNA clusters were generated on a slide using a Cluster Generation Kit (ver. 4) on an Illumina Cluster Station, according to the manufacturer's instructions. Sequencing runs for 81-mer paired-end sequence were performed using an Illumina Genome Analyzer IIx (GA IIx). The 81-mer paired-end reads were assembled (parameters k64, n51, c32.1373) using ABySS-pe v1.2.0 [50] (link). Annotation of genes from the draft genome sequences was performed using Rapid Annotation using Subsystem Technology (RAST: http://rast.nmpdr.org/ ) [51] (link) with a local manual BLASTp search. Metabolic pathway modules were predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG: http://www.genome.jp/kegg/ ) [52] (link). Genome comparison was performed using RAST, and then manually visualized by GenomeMatcher 1.69 (http://www.ige.tohoku.ac.jp/joho/gmProject/gmhome.html ) [53] (link).
8
Detecting PARE Biomarker in Plasma
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EXAMPLE 5
Detection of PARE Biomarker in Human Plasma
To determine the sensitivity of rearranged biomarkers in the presence of normal DNA, serial dilutions of tumor:normal DNA mixtures were used as templates for PCR using primers for the chromosome 4/8 translocation in Hx402. The tumor DNA dilution began at 1:125 tumor:normal and continued as a one-in-five serial dilution until reaching 1:390,625 tumor:normal mixture. PCR was performed for each of the six tumor:normal DNA mixtures and for the normal DNA control, using translocation specific primers as well as control primers from chromosome 3.
One ml of human plasma samples were obtained from patients Hx402 and Hx403 and from a control individual and DNA was purified as described (29). Whole genome amplification of plasma DNA was performed by ligation of adaptor sequences and PCR amplification with universal primers from the Illumina Genomic DNA Sample Prep Kit.
Primers designed to amplify <200 bp fragments spanning each PARE rearrangement were used in PCR from total plasma DNA using patient or control samples. Digital PCR of plasma DNA dilutions from patient Hx402 using rearrangement specific and control primers were used to quantitate the fraction mutated DNA molecules.
9
Whole-Genome Sequencing of Evolved Bacterial Strains
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Single-end 36 bp sequencing libraries were created using the Illumina Genomic DNA Sample Prep Kit according to manufacturer's instructions (5 µg input genomic DNA), and sequencing flow cells were prepared using the Illumina Standard Cluster Generation Kit. Samples were sequenced on the Illumina Genome Analyzer II, and image analysis and data extraction were performed using Illumina RTA 1.5.35.0. Reads (with qualities) were aligned to the K12 reference genome (gi|49175990|ref|NC_000913.2) using BWA v0.5.8 [147] (link) with default parameters. Whole-genome pileup files were generated using SAMtools v0.1.8–18 [148] (link) and single-nucleotide polymorphisms due to the evolution were called using custom perl scripts that compared each evolved strain with the original ancestor. Briefly, SNPs passed the filter if they were represented in at least 40% of reads in the evolved strain and at most 10% in the reference strain, with at least 5 reads covering the position in both strains. Additional heuristic filters included a confirming read from both strands, and no more than one ambiguous SNP call (“N”) or deletion (“*”) at that position. Insertions and deletions relative to the K12 reference sequence were identified using Breseq v. 0.18 with default parameters (http://barricklab.org/breseq ; [32] (link)).
10
Illumina RNA-Seq Library Preparation
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The Illumina Genomic DNA Sample Prep kit (Illumina, Inc., San Diego, CA) was used according to the manufacturer’s protocol to process double-stranded cDNA for RNA-Seq. This process included end repair, A-tailing, adapter ligation, size selection, and pre-amplification. Amplified material was loaded onto independent flow cells. Sequencing was carried out by running 36 cycles on the Illumina Genome Analyzer IIx. The quality of the RNA-Seq reads was analyzed by assessing the relationship between the quality score and error probability. These analyses were performed on Illumina RNA-Seq quality scores that were converted to phred format (http://www.phrap.com/phred/ ).
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