To confirm the bacterial species, the bacterial strain was reisolated, and its DNA was extracted according to the instructions of the Quick-DNA Universal Kit (Zymo Research—cat. N° D4068 e D4069) kit. DNA amplification was performed by 16S rDNA at a final volume of 25 μl containing all reagents needed to react in μl ultrapure sterile water, 11,3; 10 millimolar primer F, 1,5; 10 millimolar primer R, 1,5; Taq green Buffer 5x, 5,0; MgCl2, 3,0; 10 millimolar each dNTP, 1,0; Taq DNA polymerase, 0,2; DNA mold, 1,5. Genomic DNA was extracted using the Quick-DNA Universal Kit (Zymo Research) following the manufacturer’s instructions. 16S ribosomal DNA was amplified by polymerase chain reaction (PCR) using the primers P027F (5′-GAGAGTTTGATCCTGGCTAG-3′) and 1378R (5′-CGGTGTGTACSSGGCCCGGGAACG-3′) with the amplification program: 95°C for 2 min; followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 63°C for 1 min and extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The PCR products were purified and sequenced in an automated DNA ABI3730 sequencer using the primers P027F and 1378R. The sequences were aligned and edited using BioEdit 7.0.5.3 software (Hall, 1999 ) and compared to the sequences from GenBank at the NCBI (National Center for Biotechnology Information). Phylogenetic analyses were performed using MEGA 6.0 software (Tamura et al., 2013 (link)).
Quick dna universal kit
Manufactured by Zymo Research
Sourced in United States, Germany
The Quick-DNA™ Universal Kit is a lab equipment product designed for the rapid and efficient extraction of DNA from a variety of sample types, including tissues, cells, and microorganisms. The kit utilizes a simple spin-column format to isolate high-quality genomic DNA, which can be used for downstream applications such as PCR, sequencing, and various molecular biology techniques.
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Lab products found in correlation
Luminaris color probe qpcr master mix, by Thermo Fisher Scientific (4 mentions)
Ez dna methylation gold kit, by Zymo Research (3 mentions)
Hiseq 4000, by Illumina (3 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Contour hand held glucose monitor, by Bayer (2 mentions)
Cfx384 real time system, by Bio-Rad (2 mentions)
Cfx manager 3, by Bio-Rad (2 mentions)
96 well sonicator e220, by Covaris (2 mentions)
Ampure xp system, by Beckman Coulter (2 mentions)
Imagequant tl software, by GE Healthcare (2 mentions)
Ovation rrbs methyl seq system 1 16, by Tecan (2 mentions)
Epitect fast dna bisulfite kit, by Qiagen (2 mentions)
Taqman probes, by Metabion (2 mentions)
Truemethyl whole genome kit, by Tecan (2 mentions)
Accel ngs methyl seq kit, by Integrated DNA Technologies (2 mentions)
S2 focused ultrasonicator, by Covaris (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Qiamp stool kit, by Qiagen (1 mentions)
55 protocols using quick dna universal kit
1
Bacterial Species Identification by 16S rDNA
2
Culturing and DNA Extraction of T. cruzi
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T. cruzi strains Dm28c [12 (link)] and TCC [13 (link)], were used throughout this work. Epimastigotes were grown in liver infusion tryptose (LIT) medium supplemented with 10 % fetal calf serum at 28° C; total DNA was extracted using the Quick DNA Universal kit (Zymo Research).
3
Amplification of bla OXA-LIKE Genes
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Genomic DNA of strains was obtained with the Quick-DNA Universal kit (Zymo, Irvine, California, United States). blaOXA-LIKE genes were amplified by PCR using the specific primers listed in Table 1 . PCR assays were performed using the following thermocycling conditions: 94°C for 5 min; 30 cycles at 94°C for 25 s, 52°C for 40 s, and 72°C for 50 s; and a final step at 72°C for 6 min. A. baumannii ATCC®19606 was used as a positive control for blaOXA-51.
4
Epigenetic Mouse Age Estimation
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Genomic DNA was isolated from liver tissue samples from WT and HET mice using Quick DNA universal kit (Zymo Research). Evaluation of the biological age of these samples was done using the Mouse DNAge Epigenetic Aging Clock service offered by Zymo Research. This is a mouse biological age predictor based on DNA methylation levels of a small set of CpG sites [27 (link)]. This method is based on Hovrath’s aging clock [28 (link)] and uses the Simplified Whole-panel Amplification Reaction Method.
5
Genetic sampling methods for captive bovids
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Blood samples used for high-throughput sequencing were from n = 2 bongo from Paignton Zoo (UK), collected during routine vet inspection. Microsatellites were screened in n = 26 hair samples collected from captive individuals from Chester Zoo, UK (n = 5), Givskud Zoo, Denmark (n = 5), Marwell Zoo, UK (n = 3), Wolburn Safari Park, UK (n = 4), Howletts Zoo, UK (n = 7), and Knowsley Safari Park, UK (n = 2). Hair samples were collected using a sterile comb brushed firmly along the animal from neck to rump, which is then placed into a sterile sample bag. Faecal samples from waterbuck (Kobus ellipsiprymnus; n = 2 from Knowsley Safari Park, UK), and sitatunga (T. spekii; n = 1 from Parco Natura Viva, Bussolengo, Italy) were collected to test for cross-amplification of microsatellites. Samples were stored at -20 • C until use. Total genomic DNA was extracted from blood samples using DNEasy Blood and Tissue kit, following manufacturer guidelines (Qiagen, Venlo, The Netherlands). DNA from hair samples was extracted using Quick-DNA Universal kit (Zymo Research, Irvine, USA), following manufacturer guidelines, with the addition of 20 µL of 1 M dithiothreitol during lysis. DNA from faecal samples was extracted using Qiamp Stool kit following manufacturer guidelines (Qiagen). After extraction, DNA was stored at -20 • C.
6
Quantification of Global DNA Methylation
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Global DNA methylation levels were quantified using a 5-mC DNA ELISA Kit (Zymo Research) as per manufacturer’s instructions. DNA from melanoma cell lines before and after decitabine treatment was extracted by Quick-DNA Universal Kit (Zymo Research). 100 ng of genomic DNA and methylated standards were bound to an ELISA plate and methylated DNA was detected with antibodies to 5-methylcytosine, quantified by colorimetric analysis.
7
CRISPR-Cas9 Genome Editing in HCT116 Cells
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HCT116 or HCT116-derived cells (4 × 105 cells) were transfected with 2 μg each of Cas9 expression plasmid, sgRNA expression plasmid, and donor ssDNA using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). To confirm gene targeting, genomic DNA was extracted 2 days after transfection using the Quick-DNA Universal Kit (Zymo Research), and then subjected to genotyping PCR with KOD FX (Toyobo). PCR products were cloned into pCR4-TOPO (Thermo Fisher Scientific) and analyzed by DNA sequencing.
8
Gut Microbiome DNA Extraction and Amplification
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Total genomic DNA (gDNA) extraction from the entire intestine of each fish (n = 4 per treatment) was performed using the commercial Quick-DNA™ Universal Kit (ZymoResearch©). gDNA concentration was determined with a Thermo Scientific NanoDrop™ 2000c spectrophotometer (ThermoFisher Scientific©) and integrity checked by 1% agarose gel electrophoresis.
The V3-V4 region of the 16S rRNA bacterial gene was amplified by PCR from the gDNA of each sample. For PCR amplification, we used the 16S rRNA Forward primer: 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and 16S rRNA Reverse primer: 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC, which amplify a region of ~550 bp (35 (link)). PCRs were performed in a volume of 25 μL containing 12.5 μl of Dream Taq Green PCR Master Mix (2X) (Thermo Scientific©), 0.5 μL of gDNA (equivalent to 328 ± 23 ng/μl of gDNA), and 0.115 μL of each primer (125 nM). All reactions were performed in a Thermal cycler C1000 Touch™ (Bio-Rad Laboratories©) using a cycling program as follows: initial denaturation of 3 min at 95°C followed by 36 cycles of denaturation for 30 s at 95°C, annealing of 30 s at 53°C, an extension of 60 s at 72°C, and a final extension of 5 min at 72°C. PCR for no-template control was included to guarantee that no cross-tube reactions occurred.
The V3-V4 region of the 16S rRNA bacterial gene was amplified by PCR from the gDNA of each sample. For PCR amplification, we used the 16S rRNA Forward primer: 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and 16S rRNA Reverse primer: 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC, which amplify a region of ~550 bp (35 (link)). PCRs were performed in a volume of 25 μL containing 12.5 μl of Dream Taq Green PCR Master Mix (2X) (Thermo Scientific©), 0.5 μL of gDNA (equivalent to 328 ± 23 ng/μl of gDNA), and 0.115 μL of each primer (125 nM). All reactions were performed in a Thermal cycler C1000 Touch™ (Bio-Rad Laboratories©) using a cycling program as follows: initial denaturation of 3 min at 95°C followed by 36 cycles of denaturation for 30 s at 95°C, annealing of 30 s at 53°C, an extension of 60 s at 72°C, and a final extension of 5 min at 72°C. PCR for no-template control was included to guarantee that no cross-tube reactions occurred.
9
Genotyping NOS3 gene variant
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Isolation of DNA from leukocytes of peripheral blood was carried out using a set of reagents “Quick-DNAUniversalKit” (ZymoResearch, USA).
Determination of genotypes for the variant rs 61722009 of the NOS3 gene was carried out using the polymerase chain reaction method according to the previously described protocols [9 (link)].
Determination of genotypes for the variant rs 61722009 of the NOS3 gene was carried out using the polymerase chain reaction method according to the previously described protocols [9 (link)].
10
Serum Biomarkers and Glucose Dynamics
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Serum sclerostin and insulin were measured in plasma by ELISA (Alpco). Glucose levels were measured using a Bayer Contour hand‐held glucose monitor. For glucose tolerance testing, glucose (2 g/kg BW) was injected IP after a 6‐h fast. Tissues for histological analysis were collected at necropsy, weighed, and then fixed in 4% paraformaldehyde before embedding and sectioning. Adipocyte size was assessed using ImageJ. DNA was isolated from tissues using the Quick‐DNA Universal kit (Zymo Research) following proteinase K digestion.
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