DNA was extracted from the mouse blood samples containing Trypanosoma using the DNeasy DNA extraction kit (Qiagen) according to the manufacturer’s instructions. Phosphate-buffered saline was added to 150 ml of the blood sample until a final volume of 200 µl and mixed gently. Twenty microliter proteinase K was added to the solution, vortexed, and incubated at 60°C for 5 min, during which time, the tube containing the solution was inverted every 2 min. To the solution, 200 µl absolute alcohol was added, and the sample was vortexed for 10 s. The total volume of the solution was transferred to a spin column and centrifuged at 14-16,000× g for 1 min. Following centrifugation, an internal membrane was moved to the new collection column. To the DNA-binding column, 400 µl of W1 buffer was added followed by centrifugation at 14-16,000× g for 30 s. The DNA-binding column was washed by the addition of 600 µl wash buffer and centrifuged at 14-16,000× g for 3 min. The DNA was eluted by the addition of 100 µl buffer elution and recovered by centrifugation at 14-16,000× g for 1 min.
Dneasy dna extraction kit
Manufactured by Qiagen
Sourced in United States, Germany, China, Spain
The DNeasy DNA extraction kit is a laboratory product designed for the purification of DNA from a variety of sample types. It utilizes a silica-membrane-based method to efficiently capture and elute DNA, providing a reliable and consistent extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (6 mentions)
Dsdna high sensitivity kit, by Thermo Fisher Scientific (5 mentions)
Clc genomics workbench, by Qiagen (2 mentions)
Hiseq sequencer, by Illumina (2 mentions)
Dsdna high sensitivity assay kit, by Thermo Fisher Scientific (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Platinum pfx, by Thermo Fisher Scientific (1 mentions)
Platinum taq hf, by Thermo Fisher Scientific (1 mentions)
Dpni, by New England Biolabs (1 mentions)
Illustrator 2021, by Adobe (1 mentions)
Dna stool kit, by Qiagen (1 mentions)
Nextera xt dna library kit, by Illumina (1 mentions)
Nanodrop onec, by Thermo Fisher Scientific (1 mentions)
Tapestation instrument, by Agilent Technologies (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Hiseq2000 flow cell v3, by Illumina (1 mentions)
Agarose e gel, by Thermo Fisher Scientific (1 mentions)
5 mc dna elisa kit, by Zymo Research (1 mentions)
57 protocols using dneasy dna extraction kit
1
Trypanosoma DNA Extraction from Mouse Blood
2
Identification of mcr-1 and mcr-2 genes
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Total DNA was extracted by Qiagen DNeasy DNA Extraction Kit (QIAGEN, Crawley, UK) from cultures left at 37 °C overnight in Luria–Bertani (LB) media. Isolates were identified by PCR amplification and sequencing of 16S rRNA, as previously described [15 (link)]. Isolates were tested by PCR for plasmid-encoded mcr-1, using primers CLR5-F (5′-CGGTCAGTCCGTTTGTTC-3′) and CLR5-R (5-CTTGGTCGGTCTGTAGGG-3′), as previously described [8 (link)]. Similarly, isolates were tested by PCR for mcr-2, using mcr-2 full Fw (5′-ATGACATCACATCACTCTTGG-3′) and mcr-2 full Rv (5′-TTACTGGATAAATGCCGCGC-3′) as previously described [16 (link)]. Amplified DNA fragments were purified using QIAquick PCR Purification Kit (QIAGEN, Crawley, UK) and sequenced in both directions. Nucleotide and deduced amino acid sequences were analyzed and compared by BLAST, as implemented by the National Center for Biotechnology Information web site (http://blast.ncbi.nlm.nih.gov/Blast.cgi ).
3
Targeted CFTR Gene Sequencing
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Genomic DNA was isolated using DNeasy DNA extraction kit (Qiagen). A 2.6-kb PCR product was generated using Platinum pfx (Invitrogen) and primers FP-i9.1 and RP-i10.1. A second PCR was performed using sequencing labelled CFTR-NHEJFor and CFTR-NHEJRev primers to produce a 432 bp product which was subjected to next generation sequencing analysis.
Genomic DNA was isolated and treated with DpnI (NEB) for 90 min at 37 °C to restrict plasmid DNA. A 2.6-kb PCR product was generated using Platinum Taq HF (Invitrogen) and primers 5′-AATTTTGTAAATTTGTTTCATC-3′ and 5′-ACTTGCTTTGCCATTAACAGA-3′. 435-bp amplicons for sequencing by GS FLX++ chemistry (Eurofins Genomics), were generated with the primers 5′-ATCATGTGCCCCTTCTCTGT-3′ and 5′-CGTAGACTAGTGCTTTGATGACGCTTCTGTAT-3′ tagged with a unique 10 nucleotide multiplex-identifier (MID). Sequence alignments were performed using Clustal W42 (link) and MegAlign (Version 11.2.1. DNASTAR. Madison, Wisconsin).
Genomic DNA was isolated and treated with DpnI (NEB) for 90 min at 37 °C to restrict plasmid DNA. A 2.6-kb PCR product was generated using Platinum Taq HF (Invitrogen) and primers 5′-AATTTTGTAAATTTGTTTCATC-3′ and 5′-ACTTGCTTTGCCATTAACAGA-3′. 435-bp amplicons for sequencing by GS FLX++ chemistry (Eurofins Genomics), were generated with the primers 5′-ATCATGTGCCCCTTCTCTGT-3′ and 5′-CGTAGACTAGTGCTTTGATGACGCTTCTGTAT-3′ tagged with a unique 10 nucleotide multiplex-identifier (MID). Sequence alignments were performed using Clustal W42 (link) and MegAlign (Version 11.2.1. DNASTAR. Madison, Wisconsin).
4
Mitogenome Sequencing of Centrotinae
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For sequencing mitogenomes, we used DNeasy DNA Extraction Kit (Qiagen) to extract the total genomic DNA from thoracic muscle tissues. The NGS (Illumina HiSeq X; Biomarker Tech, Beijing, China) was employed to determine the four mitogenomes of Centrotinae. A total of 16,902,362/13,815,488/20,016,944/13,564,230 clean paired reads, then assembled using Geneious 9.0.2 [24 (link)] with the mitogenomes of Leptobelus gazella (JF801955) and Tricentrus brunneus (MK746138) were employed as references. The annotation of the mitogenomes was performed using Geneious 9.0.2. Furthermore, the MITOS Web Server (Leipzig, Germany) [25 (link)], with the invertebrate mitochondrial genetic code (transl_table = 5), was made a forecast for the position and secondary structure of the tRNA, and Adobe Illustrator 2021 was employed to draw manually as the predicted results show. The PCGs boundaries were recognized by the open reading frames (ORFs) employing translation table 5 and alignment with homologous reference sequences was performed in Geneious 9.0.2. In addition, CGView Server (http://cgview.ca/ (accessed on 26 June 2021)) [26 (link)] was used to generate the mitogenome maps online.
5
DNA Extraction from Blood and Feces
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DNA from blood stains was extracted with DNeasy DNA Extraction Kit (Qiagen) according to the producer’s manual. DNA from faeces was extracted with the DNA Stool Kit (Qiagen) according to the manufacturer’s instructions modified according to Segelbacher [25 ] and Regnaut et al. [26 ].
6
Isolation and Characterization of T. palmi
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The specimens of T. palmi were collected from the Odisha state of India from Eggplant (Solanum melongena). The specimens were morphologically identified by the second author (K.T) with the available taxonomic keys [9 ], and preserved in absolute ethyl alcohol at −80°C in Centre for DNA Taxonomy, Molecular Systematics Division, Zoological Survey of India, Kolkata. The DNeasy DNA Extraction kit (Qiagen, Valencia, CA) was used to extract the genomic DNA and the concentration was measured on a quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) using a dsDNA high-sensitivity kit with the standard protocol.
7
Whole Genome Sequencing of Bacterial Isolates
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We performed whole genome sequencing on all isolates tested in this study. Total DNA was extracted from an overnight pure culture in 5 mL of tryptic soy broth using the DNeasy DNA extraction kit (Qiagen, Valencia, CA). DNA quality was verified using NanoDrop OneC (ThermoFisher, Waltham, MA) before quantification using a Qubit fluorometer and dsDNA high-sensitivity assay kit (ThermoFisher). Sequencing libraries were generated using the Nextera XT DNA library kit (Illumina, San Diego, CA). Quality and quantity of each sample library was measured on a TapeStation instrument (Agilent Technologies, Santa Clara, CA). The genomes were sequenced as 2 × 100 bp or 250 bp reads on an Illumina HiSeq sequencer according to the manufacture's specifications, with a minimum depth of coverage of 30×. Sequence reads were assembled de novo using CLC Genomics Workbench (CLC Bio, Cambridge, MA). We used the Center for Genomic Epidemiology pipeline to obtain confirmation of species identification and sequence type. Clonal complexes (CCs) were determined using the MLST data by the goeBURST algorithm (http://www.phyloviz.net/goeburst/ ).
8
Mitogenome Sequencing of Beetle Genera
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A total of 17 species of these three genera were selected for whole mitogenome sequencing (Additional file 1 : Table S1). All the specimens were stored in 95% ethanol prior at − 20 ℃ in Chongqing Normal University (CQNU). Total DNA was extracted from the muscle tissues of thorax using the DNeasy DNA Extraction kit (QIAGEN Shanghai, China). The concentration of double-stranded DNA (dsDNA) in extraction was assayed on a Qubit fluorometer using a dsDNA high-sensitivity kit (Invitrogen Shanghai, China).
9
Whole Genome Sequencing of High-Persister Mutants
10
De novo Chloroplast Genome Sequencing of Adenium obesum
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The fresh leaves of A. obesum were collected from the wild condition of desert habitat near to Riyadh, Saudi Arabia. The total genomic DNA was isolated using QIAGEN DNeasy DNA extraction kit. The de novo sequencing base calling was performed using the Illumina Pipeline 1.3.2 (Nie et al., 2012 ). The raw reads were filtered using FastQC to obtain the high-quality clean data by removing adaptor sequences using trimmomatic and low-quality reads with Q-value ≤ 20. The filtered reads were assembled using Spades (Bankevich et al., 2012 (link)), and annotated using GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html ) (Tillich et al., 2017 (link), Hansen et al., 2007 ). Further downstream analysis from the assembled cp genome included the repeat structure (Benson, 1999 (link), Timme et al., 2007 ) and small inversion (Nagano et al., 1991 (link), Yang et al., 2010 , Doorduin et al., 2011 (link), Castro et al., 2013 (link), Beier et al., 2017 ).
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