Dermacentor ticks were collected from the environment (questing) and off domestic animals from 15 aimags across Mongolia in 2019 (Uvs, Khovd, Govi-Altai, Zavhan, Khuvsgul, Arkhangai, Bayankhongor, Arkhangai, Uvurkhangai, Bulgan, Tuv, Dundgovi, Khentii, Dornogovi, Sukhbaatar, and Dornod; Figure 1 ). Adult ticks were morphologically identified as D. nuttalli or to the genus level as Dermacentor spp. by entomologists using local keys (Boldbaatar and Byambaa, 2015 ). In total, 7,275 ticks were collected and sorted into 1,489 pools according to location and collection source (environment vs. animal). Of these pools, 377 pools of adult stage ticks, representing pools from all sampled provinces, were selected for analysis by next-generation sequencing, including 51 pools collected from livestock (Tables 1 , 2 ). Whole ticks in 250 μl of ATL buffer were punctured with a fine tip under a stereomicroscope to release the tissue from the hard chitin exoskeleton prior to adding 2 mg/ml of Proteinase K solution. Samples were then incubated at 55°C overnight. A total volume of 250 μl homogenized solution was then used for DNA extraction on the QIAsymphony® SP instrument with QIAsymphony® DSP DNA Mini Kit using Tissue LC 200 DSP protocol (Qiagen, Hombrechtikon, Switzerland). The DNA was eluted in 50 μl of ATE buffer and stored at −20°C until use.
Qiasymphony dsp dna mini kit
Manufactured by Qiagen
Sourced in Germany, United States, Switzerland
The QIAsymphony DSP DNA Mini Kit is a nucleic acid purification kit designed for the automated extraction of DNA from a variety of sample types using the QIAsymphony system. The kit utilizes magnetic bead technology to efficiently capture and purify DNA, providing high-quality nucleic acid for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Qiasymphony sp, by Qiagen (5 mentions)
Nextseq 500, by Illumina (5 mentions)
Glomax 96 microplate luminometer, by Promega (4 mentions)
Chelex 100 resin, by Bio-Rad (4 mentions)
Proteinase k, by Merck Group (4 mentions)
Hiseq 2500, by Illumina (4 mentions)
Hiseq x ten, by Illumina (3 mentions)
Nextera xt kit, by Illumina (3 mentions)
Nextera xt dna library prep kit, by Illumina (3 mentions)
Tissue lc 200 dsp protocol, by Qiagen (2 mentions)
Truseq nano dna library prep kit, by Illumina (2 mentions)
Qiaamp dna blood mini qiacube kit, by Qiagen (2 mentions)
Qiasymphony sp automated instrument, by Qiagen (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Allprep dna rna mirna universal kit, by Qiagen (2 mentions)
Rotor gene q, by Qiagen (2 mentions)
Deparaffinization solution, by Qiagen (2 mentions)
Qiasymphony sp system, by Qiagen (2 mentions)
Nextseq 500 mid output v2 kit 300 cycles, by Illumina (2 mentions)
Nextseq 500 mid output kit, by Illumina (2 mentions)
58 protocols using qiasymphony dsp dna mini kit
1
Tick Collection and Identification in Mongolia
2
Organoid DNA Extraction and Sequencing
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Organoids were dissociated and DNA was isolated using the QiaSymphony DSP DNA mini kit (Qiagen, cat. No. 937236). Libraries were prepared using the Truseq DNA nano library prep kit (Illumina, cat. No. 20015964). Paired-end sequencing was performed (2 × 150 bp) on the generated libraries with 30x coverage using the Illumina HiSeq Xten at the Hartwig Medical Foundation.
3
Automated DNA Extraction from Blood
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DNA was extracted from EDTA blood or from dry blood spots in filter cards (CentoCard). We used two automated procedures; the spin-column based extraction was performed on QIAcube instrument with QIAamp DNA Blood Mini QIAcube Kit (Qiagen, Valencia, CA, USA) following the manufacturer instructions. Alternatively, the QIAsymphony DSP DNA Mini Kit (Qiagen) on the QIAsymphony instrument was used to purify the DNA from blood. Following extraction all DNA samples were stored at −20 °C. Before the analysis the DNA quality and concentration was determined photometrically (OD260/OD280 1.8–2.0).
4
DNA Extraction and Sequencing of Pneumococcus
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Bacterial growth was harvested from the agar plates and pre-lysed by the recommended method for Gram-negative bacteria (Qiagen, Manchester, UK), which is effective for pneumococcus and closely related streptococci. DNA was extracted from the lysates using a QIAsymphony SP automated instrument and a QIAsymphony DSP DNA Mini Kit (Qiagen), using the tissue extraction protocol. DNA concentrations were measured using the Quant-IT Broad Range dsDNA Kit (Life Technologies, Paisley, UK) and GloMax® 96 Microplate Luminometer (Promega, Southampton, UK). WGS was performed using Illumina methodology by the PHE Genomic Services Delivery Unit (GSDU, Colindale, UK). The resulting data were automatically analysed using an in-house bioinformatics pipeline for S. pneumoniae.
5
Salmonella Enteritidis Genome Sequencing
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Salmonella enterica serovar Enteritidis strains included in this study were isolated from clinical (n = 206) or environmental samples (n = 11) as outlined in S1 Table . DNA was extracted from isolates grown overnight at 37°C on horse blood agar, using the QiaSymphony DSP DNA Mini kit (Qiagen) according to the manufacturer’s protocol. DNA was prepared for sequencing using the Nextera XT kit (Illumina) and sequenced on the NextSeq500 using the NextSeq 500 Mid Output v2 kit (300 cycles) (Illumina) according to the manufacturer’s instructions. Raw sequence files and associated metadata have been submitted to the European Nucleotide Archive with project accession number PRJEB22598.
6
Comprehensive DNA Isolation and Sequencing Protocol
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DNA from fresh frozen tumor tissue was isolated using the Allprep DNA/RNA/miRNA Universal Kit (Qiagen) or QIAamp DNA mini (QIAGEN). DNA from formalin fixed paraffin embedded tissue was isolated using the GeneRead DNA FFPE Kit (QIAGEN). DNA from peripheral blood was isolated using QIAamp DNA Blood Mini (Qiagen) or QIASymphony DSP DNA Mini Kit (Qiagen). The isolation process was followed by quality control and quantification using a Qubit 2.0 Fluorometer (Invitrogen) and a TapeStation 2200 system (Agilent). Libraries for whole-genome sequencing were prepared with the Illumina TruSeq Nano (100 ng genomic DNA as input). Both tumor and control (germline) samples were sequenced on 2 lanes Illumina HiSeq X Ten (Supplementary Data 15 ). Libraries for whole-exome sequencing were prepared with the Agilent SureSelect All Exon Kit v5 or v5 + UTRs (200 ng input). The libraries were sequenced on Illumina HiSeq 2000, HiSeq 2500 or HiSeq 4000 (Supplementary Data 15 ). Samples were processed centrally by the NCT Molecular Precision Oncology Program Sample Processing Laboratory (SPL) and sequenced by the DKFZ Genomics and Proteomics Core Facility (GPCF). Further information and exceptions are listed in Supplementary Data 8 .
7
DNA Extraction from Hair and Blood Samples
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Deoxyribonucleic acid was prepared from the hair roots using a standard hair-preparation procedure. Briefly, 186 μL Chelex 100 Resin (Bio-Rad Laboratories, Hercules, CA) and 14 μL of proteinase K (20 mg/mL; Merck KgaA, Darmstadt, Germany) were added to the sample. The mix was incubated at 56°C for 2 h and the proteinase K was inactivated for 10 min at 95°C. For DNA preparation from blood samples, 200 μL of blood was used and isolated on the Qiasymphony instrument using the Qiasymphony DSP DNA mini kit (Qiagen, Hilden, Germany). Samples for two horses failed to meet the DNA quality requirements for genotyping and were replaced (Fig 1 ). Descriptive statistics of the final horses selected for genotyping are shown in Table 2 . The final horses selected represented 107 sires and 226 dams.
8
Multiplex PCR for Enteric Pathogens
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DNA was extracted using the QIAsymphony DSP DNA Mini Kit (Qiagen). All specimens were spiked with 10 µl of modified green fluorescent protein E. coli,18 (link) which acted as an internal positive control to minimise the risk of false negative reporting. Eluted DNA extracts were used to detect a range of BEPs using real-time polymerase chain reaction (PCR) primers and probes on a Rotor-Gene Q (Qiagen). The multiplex PCR assay included gene targets for Shigella spp./enteroinvasive E. coli, Campylobacter jejuni/coli, Salmonella spp., Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), and enteropathogenic E. coli (EPEC). The amplification parameters were 95 °C for 5 min, followed by 95 °C for 15 s and 60 °C for 60 s (40 cycles). The cycle threshold was set at 0.05 for all targets. In a secondary analysis, real-time PCR using the Applied Biosystems TaqMan 7500 (Thermo Fisher Scientific) was used to detect the presence of mphA, an AMR gene associated with resistance to the macrolide, azithromycin.19 (link) The PCR assay was performed using all eluted DNA extracts which returned a positive result for one of the BEP target genes, and for comparison, a random subset of 100 DNA extracts which returned a negative result for all BEP gene targets. Details of all the primers, probes and gene targets are provided in Supplementary Table 1.
9
DNA Extraction from Hair and Blood
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DNA was extracted from hair roots using a standard procedure of hair preparation. Briefly, 186 μL of Chelex 100 Resin (Bio-Rad Laboratories, Hercules, CA) and 14 μL of proteinase K (20 mg/mL; Merck KgaA, Darmstadt, Germany) were added to each sample. This mix was incubated at 56 °C for 2 h and proteinase K was inactivated for 10 min at 95 °C. For DNA preparation from blood, DNA from 350-μL blood samples were extracted by using the Qiasymphony instrument and the Qiasymphony DSP DNA mini kit (Qiagen, Hilden, Germany).
10
Genomic DNA Extraction and Clinical Exome Sequencing
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Genomic DNA was isolated from peripheral blood using the QIAsymphony DSP DNA Mini Kit (Qiagen, Hilden, Germany) following manufacturer's instructions. Library preparation and clinical exome capture were performed using the Twist Custom Panel kit (Twist Bioscience, San Francisco, CA, USA) and sequenced on the NovaSeq 6000 platform (Illumina). The BaseSpace pipeline (Illumina) and the TGex software (LifeMap Sciences) were used for variant calling and annotation. Reads were aligned to human genome build GRCh37/hg19. Based on the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP), a minimum depth coverage of 20X was considered suitable for analysis. The mean coverage for the target region was 152X.
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