Faeces suspended in RNAlater reagent (Ambion, Life Technologies, Carlsbad, CA) were subjected to DNA extraction using a High Pure Viral Nucleic Acid kit (Roche Diagnostics GmbH, Mannheim, Germany) or QIAamp Viral RNA Mini Kit (Qiagen, Valencia CA). DNA from spleen tissues was prepared using DNAzol reagent (Molecular Research Center, Cincinnati, OH) or QIAamp DNA Mini kit (Qiagen). For nested PCR screen targeting for NS1 gene of BuV, we followed the method of Sasaki et al.9 (link).
Dnazol reagent
Manufactured by Molecular Research Center
Sourced in United States
DNAzol reagent is a guanidinium-detergent based solution designed for the isolation and purification of DNA from various biological samples. It functions by efficiently lysing cells and solubilizing cellular components, while preserving the integrity of the DNA.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna mini kit, by Qiagen (1 mentions)
Rnalater reagent, by Thermo Fisher Scientific (1 mentions)
High pure viral nucleic acid kit, by Roche (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Take3 micro volume plate, by Agilent Technologies (1 mentions)
Gen5 microplate reader, by Agilent Technologies (1 mentions)
Biosprint 96 dna plant kit, by Qiagen (1 mentions)
Accuspin micor 17, by Thermo Fisher Scientific (1 mentions)
8 oh dg elisa kit, by Cayman Chemical (1 mentions)
10 protocols using dnazol reagent
1
BuV DNA Extraction and Detection
2
DNA Extraction from Frozen Tissues
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The UC frozen tissues were lysed with 1.0 mL DNAzol reagent (Molecular Research Center, Cincinnati, OH, USA) and 200 μg/mL proteinase K (Sigma-Aldrich, St. Louis, MO, USA) by gentle pipetting. A 0.5-mL aliquot of chloroform was added to the cell lysate. This mixture was shaken vigorously and was then centrifuged at 12,000 rpm for 5 min. After centrifugation, the supernatant and the DNA pellet were collected. The DNA was dissolved in 50 μL of 8 mM NaOH and was quantified with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) for the determination of double-stranded DNA (For details, see the online Supplementary MATERIALS AND METHODS ).
3
Genomic DNA Extraction from Plant Leaves
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DNA was extracted from lines in the A-set and V-set for GBS using BioSprint 96 DNA Plant Kits (Qiagen, Hombrechtikon, Switzerland) from 2 to 3 leaves of 2-weeks-old seedlings. For the SSR marker test, DNA from a bulk of six leaves of 5 days old plants was extracted using DNAzol Reagent (Molecular Research Center, Inc. Technical Bulletin 6). The tissue was ground using liquid nitrogen then 300 μl DNAzol Reagent was added to this powder and left for 5 min at the room temperature. A volume of 300 μl chloroform was added to the previous mix and left at the room temperature for 5 min before it was centrifuged using Fisher Scientific accuSpin Micor 17 centrifuge (Loughborough, United Kingdom) at 12000 × g for 10 min. The washing process was done in three steps by adding three different washing solutions as follows: (1) absolute alcohol, (2) DNAzol + 75% Ethanol and (3) 75% alcohol only. All genotypes were centrifuged using the Fisher Scientific accuSpin Micor 17 centrifuge for 4 min at 5000 × g after each washing step. The extracted DNA was then re-suspended in 150 μl of TE buffer. DNA concentration was measured using spectrophotometry (Gen5TM microplate reader and image software with Take3TM micro-volume plates [BioTek, Winooski, VT, United States]).
4
Molecular Detection of Plasmodium falciparum
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After each behavioral assay, all females exposed to infectious blood meal and a subset of females exposed to non-infectious blood were tested for their infection status. They were conserved and stored individually in 100 µl of DNAzol® reagent (Molecular Research Center, Inc, Cincinnati, OH, USA). The entire insect was used for females killed 6–8 dpbm. Only the cephalothoraxes were used for females killed 12–14 dpbm; this allows to detect infected mosquitoes at days 6 to 8 and exclusively the presence of sporozoites stages at days 12 to 14. DNA extraction from individual mosquitoes was performed using DNAzol® according to the manufacturer’s instructions. P. falciparum detection was then carried out by qPCR50 (link),51 (link). Females were considered positive for P. falciparum when the Cq (quantification cycle) ranged from 25 to 35 and when the Tm (primer melting temperature) ranged from 75.5 to 80.
5
Bladder Cancer Tissue Sample DNA Extraction
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Freshly frozen samples from 7 bladder cancer tissues were used for this study. The samples were obtained from Tampere University Hospital and include five urothelial carcinomas, one lymphoepithelial carcinoma and one undifferentiated carcinoma. DNA was extracted using DNAzol reagent (Molecular Research Center, Inc. Cincinnati, OH), according to manufacturer’s protocol. The use of the clinical samples was approved by the ethical committee of the Tampere University Hospital.
6
Mitochondrial DNA Copy Number Analysis
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A PCR-based analysis of the mitochondrial DNA copy number was conducted as described earlier [67 (link)]. DNA was extracted from 45 mg of frozen liver tissue using DNAzol reagent (Molecular Research Center, Inc., Cincinnati, OH), as per the manufacturer’s instructions. For this analysis, a gene encoded by mitochondrial DNA (CYTB) was compared to a gene encoded by genomic DNA using GAPDH primers [67 (link)]. Samples were analyzed using SYBRgreen-based PCR on the ABI StepOnePlus™ Real-Time PCR System using the cycling conditions described earlier [68 (link)]. A melt curve analysis was performed at the end of the amplification. Genes were compared using the following equations: ΔCT = (nucDNA CT − mtDNA CT) and relative mitochondrial DNA content was estimated as 2 × 2ΔCT. The reactions were carried out in triplicates.
7
Assessing Placental and Fetal Oxidative Damage
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The levels of 8-hydroxydeoxyguanosine (8-OHdG) were assessed in the placental and fetal DNA to evaluate the oxidative damage produced by the UFP. The DNAzol reagent (Molecular Research Center, Cincinnati, OH, USA) was used to isolate genomic DNA of whole placentas and fetuses (as previously described). An 8-OH-dG ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) was used, and all procedures were conducted according to the manufacturer’s instructions.
8
Knock-in of Genes in HEK293FT Cells
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For the knock-in experiments, 200 ng of Cas9+sgRNA vector and 400 ng of an oligonucleotide donor DNA were transfected to HEK293FT cells stably expressing GFP1-10 per 24-well plate (Eppendorf). Genomic DNA was extracted from cells 3 days after transfection with the DNAzol reagent (Molecular Research Center, Inc). We further enriched GFP-positive cells by fluorescence-activated cell sorting.
9
Quantifying BAd-ΔE1E3 Virus Recovery
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Purified BAd-ΔE1E3 particles at various dilutions (0, 5 × 101, 5 × 103, 5 × 105, or 5 × 107 p.f.u.) in 50 μL were spiked in the strips of PUF of the ASAP system. Virus was recovered by adding 500 μl PBS to the PUFs and squeezing the liquid out of the PUFs by centrifugation. Un-spiked virus samples served as controls for the determination of virus recovery from the PUFs. DNA was isolated using the DNAzol reagent (Molecular Research Center). The isolated DNA was resuspended in 25 μL of distilled water, and the amounts of extracted nucleic acid were quantified by a qPCR assay using known amounts of purified BAd-ΔE1E3 nucleic acid as standards.
The efficiency of virus recovery was calculated as –
The efficiency of virus recovery was calculated as –
10
Extracting DNA from Pseudocorynosoma Parasites
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Eight specimens of Pseudocorynosoma sp., from ruddy duck, seven of P. anatarium from bufflehead duck and 15 specimens of P. constrictum from waterfowl species were placed individually in tubes and digested overnight at 56C in a solution containing 10 mM Tris-HCl (pH 7.6), 20 mM NaCl, 100 mM Na 2 EDTA (pH 8.0), 1% Sarkosyl, and 0.1 mg/ml proteinase K. Following digestion, DNA was extracted from the supernatant using the DNAzol reagent (Molecular Research Center, Cincinnati, Ohio) according to the manufacturer's instructions.
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