DNA from the contemporary samples was extracted using the E.Z.N.A. Tissue DNA Extraction kit following the manufacturer's instructions (OMEGA Bio-Tek, CA, USA). DNA from the historical samples was extracted using the MicroElute Genomic DNA kit (OMEGA Bio-Tek, CA, USA). To minimize contamination, DNA-extraction from historical samples was conducted in a dedicated clean laboratory facility in a separate building with restricted access and with no extraction of contemporary DNA samples taking place. One or two otoliths and scales (if available) were incubated in a 55C shaking water bath for approximately 24 hours to maximize lysis. Final DNA elution was conducted using a small amount of elution buffer (10-15 µL) in order to maximize DNA concentration. The quality of DNA in the historical samples was tested by polymerase chain reaction (PCR) amplification of the Cryptochrome2b.2 gene (O'Malley et al. 2010) using an annealing temperature of 55C. Negative controls were included in the PCR amplifications in order to control for extraneous DNA contamination.
Microelute genomic dna kit
Manufactured by Omega Bio-Tek
Sourced in United States
The MicroElute Genomic DNA Kit is a product from Omega Bio-Tek designed for the rapid and efficient extraction of genomic DNA from a variety of sample types. The kit utilizes a silica-based membrane technology to capture and purify DNA, providing a simple and reliable method for DNA isolation.
Automatically generated - may contain errors
Lab products found in correlation
Monarch pcr dna cleanup kit, by New England Biolabs (3 mentions)
Phusion polymerase, by Thermo Fisher Scientific (2 mentions)
E z n a, by Omega Bio-Tek (2 mentions)
Ezna tissue dna extraction kit, by Omega Bio-Tek (1 mentions)
Miseq pe300 platform novaseq pe250 platform, by Illumina (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Quantus fluorometer, by Promega (1 mentions)
Nucleospin 96 tissue kit, by Macherey-Nagel (1 mentions)
Phusion polymerase, by New England Biolabs (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Perfectstart green qpcr supermix, by Transgene (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Ds 11 fx, by DeNovix (1 mentions)
Genemapper software, by Thermo Fisher Scientific (1 mentions)
Genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 7 flex, by Thermo Fisher Scientific (1 mentions)
Glomax multi, by Promega (1 mentions)
17 protocols using microelute genomic dna kit
1
Historical and Contemporary DNA Extraction
2
Comprehensive Gut Microbiome Profiling
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Fresh rectal feces were collected from different groups of animals and frozen in liquid nitrogen till analysis. Gut microbiota was analyzed using 16S rDNA sequencing as described previously (14 (link)). Briefly, DNA was extracted from the feces samples using a Micro Elute Genomic DNA Kit (Omega Bio-Tek, Norcross, GA, United States). The V3-V4 region of bacterial 16S rRNA was amplified by using the primers: 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR product was then extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States) according to the manufacturer’s instructions and quantified using Quantus™ Fluorometer (Promega, United States). Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, United States) according to the standard protocols by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Sequences were assigned to operational taxonomic units (OTUs) at 97% similarity.
3
Echinococcus multilocularis Specimen Collection
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Echinococcus multilocularis specimens were collected from gastrointestinal (GI) tracts of red foxes and coyotes of either road-killed or trap-harvested animals (trapped for purposes independent of this study), collected between 2012 and 2017 in Western Canada. Trapped animals were obtained from licensed trappers with the collaboration of the Alberta Trappers Association. GI tracts were screened using a modification of the scraping, filtration and counting technique, to identify and collect Echinococcus spp. worms [35 (link),36 (link)]. We analysed Em worms from 70 coyotes and 13 foxes from northern, central and southern Alberta (AB); four coyotes from north-west British Columbia (BC); and 10 coyotes from southeast Saskatchewan (SK). Extraction of DNA was performed on up to five individual worms per host using the Nucleospin 96 Tissue Kit (Macherey-Nagel, Germany) for samples processed in France (Anses Nancy Laboratory for Rabies and Wildlife) and the E.Z.N.A. MicroElute Genomic DNA Kit (Omega Bio-tek, US) for samples processed in Canada (University of Calgary, Faculty of Veterinary Medicine). Extraction was performed following the manufacturer's instructions, and DNA was stored at −20°C until processed.
4
DNA Extraction from Stool and Colon
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DNA from stool samples and normal colonic mucosal tissue samples, most of which were collected from the same volunteers, was extracted using the Micro Elute Genomic DNA kit (D3096-01; Omega Bio-Tek, Inc., Norcross, GA, USA) according to the manufacturer's instructions. The reagent used to isolate DNA from trace amounts of sample was effective for the preparation of DNA of the majority of bacteria. Sample blanks consisted of unused swabs processed through DNA extraction and were tested to contain no 16S amplicons. The total DNA was eluted in 50 µl of elution buffer by modification of the procedure described by manufacturer (Qiagen, Hilden, Germany) and stored at −80°C until measurement in the PCR by LC-Bio (Hangzhou, China).
5
Quantifying CRISPR Editing Efficiency
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Nucleofected cells were harvested at indicated timepoints and genomic DNA was extracted (MicroElute Genomic DNA Kit, Omega Bio-Tek). Genomic regions around CRISPR target sites were PCR amplified using Phusion polymerase (New England BioLabs) and primers located at least 250 bp outside of sgRNA cut sites. After size verification by agaorse gel electrophoresis, PCR products were column-purified (Monarch PCR & DNA Clean-up Kit, New England BioLabs) and submitted for Sanger sequencing (Genewiz) using unique sequencing primers. The resulting trace files for edited cells versus control cells (nucleofected with non-targeting Cas9:sgRNA RNPs) were analyzed for predicted indel composition using the Inference of CRISPR Edits (ICE) web tool [24 ]. Indel percentage is defined in the ICE analysis as the editing efficiency (percentage of the pool with non-wild type sequence) as determined by comparing the edited trace to the control trace. Knockout percentage represents the proportion of cells that have either a frameshift or 21+ bp indel.
6
Genomic DNA Extraction and 18S rRNA Sequencing of S. fuscescens
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Total genomic DNA were extracted from cultured RFF cells (at passage 30) or fin tissues of S. fuscescens using microElute Genomic DNA kit (Omega Bio-tek, Norcross, GA). The quantitation and quality of isolated DNA was tested by NanoDrop™ 2000 spectrophotometers (Thermo Fisher Scientific). Fragments of S. fuscescens 18S ribosomal RNA (~700 bp) were amplified using primers 3′-CCA TTG GTT CAT TCG GAG TA-5′ and 3′ TTG GAT GGT TTA GTG AGG TC-5’. The PCR products were sequenced, and the obtained sequences were aligned against known S. fuscescens 18S rRNA sequences from NCBI database (GenBank Accession No. AB276986.1 ).
7
RNA Isolation and qRT-PCR Analysis
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TRIzol reagent (Tiangen, Beijing, China; DF424) was used to isolate total RNA from tissues and cells, and the complementary DNA reverse transcription kit was used to create complementary DNA (Takara Bio, San Jose, CA; RR037A). PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing, China) was used for the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) (AQ601). For normalization, GAPDH was used as an internal control. The 2−ΔΔCT method was used to determine the relative expression of the target gene. Table 2 lists the primers used in this study for qRT-PCR. MicroElute Genomic DNA Kit (Omega Bio-tek, Norcross, GA; D3396) was used to extract genomic DNA according to the manufacturer’s instructions. Table 2 contains a list of the PCR primers.
8
DNA Extraction from Umbilical Cord
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Umbilical cords were cut to a mung bean size (approximately 30 μg), and a MicroElute Genomic DNA Kit (OMEGA Bio-Tek, Inc., Norcross, GA, USA) was used for DNA extraction according to the manufacturer’s specifications. The optical density (DNA absorbance ratio) and concentration of the extracted DNA were measured using a Nano Q microspectrophotometer (BoAo, USA). Then, 1% agar gel electrophoresis was performed to test DNA integrity. The DNA solutions were stored at −20 °C until use.
9
CRISPR Indel Composition Analysis
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Nucleofected cells were harvested at indicated time points and genomic DNA was extracted (MicroElute Genomic DNA Kit, Omega Bio-Tek). Genomic regions around CRISPR target sites were PCR amplified using Phusion polymerase (Thermo Fisher Scientific) and primers located (whenever possible) at least 250 bp outside sgRNA cut sites. After size verification by agaorse gel electrophoresis, PCR products were column purified (Monarch PCR & DNA Clean-up Kit, New England Biolabs) and submitted for Sanger sequencing (Genewiz) using unique sequencing primers. The resulting trace files for edited cells versus control cells (nucleofected with nontargeting Cas9:sgRNA RNPs) were analyzed for predicted indel composition using the Inference of CRISPR Edits web tool (32 (link)). See detailed CRISPR RNP nucleofection protocol in Supplementary data for general PCR conditions used for Phusion polymerase. See Supplementary Table S3 for a list of all PCR and sequencing primers used, as well as PCR conditions specific to particular genomic regions.
10
Genomic DNA Extraction from Cells
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Total cellular DNA was purified using E.Z.N.A. MicroElute Genomic DNA Kit (Omega Bio-tek, Norcross, GA, USA). In brief, at the indicated time points, the cells were washed twice, scraped from the plates, centrifuged at 1000 × g, suspended in PBS (Sigma-Aldrich) and processed according to the manufacturer’s protocol. The purity (absorbance ratio at 260/280) and concentration of DNA samples were determined spectroscopically using DeNovix DS-11 FX+ (DeNovix Inc., Wilmington, DE, USA).
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