Total genomic DNA was extracted using the Qiagen QIAamp DNA Mini Kit 50 according to the manufacturer's instructions. The 16S rRNA gene was amplified using two universal primers, namely, 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1406R (5′-GACGGGCGGTGTGTRCA-3′) (Baker and Cowan, 2004 (link); Devereux and Wilkinson, 2004 (link)). The reaction mixture and PCR reaction parameters were set up according to earlier research (Najar et al., 2018 (link)). The Applied Biosystems' BigDyeTMTerminator version 3.1 cycle sequencing kit was used to sequence the samples on an AB3500 Genetic Analyzer. The Clustal W software was used to match the 16S rRNA sequences with representative sequences from similar taxa (Thompson et al., 1994 (link)). Using the MEGA 11 and FigTree software, a phylogenetic tree was created using the maximum likelihood method (Tamura et al., 2021 (link)) and Jukes-Cantor evolutionary distance measurement (Erickson, 2010 (link)).
Qiaamp dna mini kit 50
Manufactured by Qiagen
Sourced in Germany
The QIAamp DNA Mini Kit 50 is a DNA extraction and purification kit designed for the isolation of genomic DNA from a variety of sample types, including blood, tissue, and cells. The kit utilizes a silica-based membrane technology to efficiently capture and purify DNA, making it suitable for downstream applications such as PCR, sequencing, and other molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bigdyetmterminator version 3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Ab 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Ez dna methylation gold kit 200, by Zymo Research (1 mentions)
Abi3130 automatic sequencer, by Thermo Fisher Scientific (1 mentions)
Hot firepol blend master mix, by Solis BioDyne (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
13 protocols using qiaamp dna mini kit 50
1
Phylogenetic Analysis of Microbial 16S rRNA
2
Genomic DNA Extraction and Bisulfite Conversion
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QIAamp DNA Mini Kit (50) (51304; Qiagen, Hilden, Germany) was used to extract the genomic DNA of the human normal liver cell line and the five human HCC cell lines according to the instructions. Thereafter, the genomic DNA was processed by sodium bisulfite: 2 μg DNA was dissolved in 50 μL deionized water, denatured in 0.2 mol·L−1 NaOH at 50 °C for 10 min and water bathed in fresh 30 μL 10 mmol·L−1 hydroquinone and 520 μL sodium bisulfite (3.6 mol·L−1, pH 5.0) at 50 °C for 18 h. After being purified in adsorption column, it was precipitated in absolute alcohol and dissolved in 50 μL deionized water.
3
Tokara Horse DNA Extraction Protocol
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In 2016, 123 Tokara horses were registered by the JEAA. Blood samples were collected from
each of the 123 horses (24 horses [Nos. 1–24] in Nakanoshima, 57 [Nos. 25–81] in
Kaimondake, 42 [Nos. 82–123] in Iriki) using EDTA as an anticoagulant. Genomic DNA was
extracted using a QIAamp® DNA Mini Kit (50) (QIAGEN GmbH, Hilden, Germany) according to
the manufacturer’s protocol.
each of the 123 horses (24 horses [Nos. 1–24] in Nakanoshima, 57 [Nos. 25–81] in
Kaimondake, 42 [Nos. 82–123] in Iriki) using EDTA as an anticoagulant. Genomic DNA was
extracted using a QIAamp® DNA Mini Kit (50) (QIAGEN GmbH, Hilden, Germany) according to
the manufacturer’s protocol.
4
Genetic Profiling of Pseudomonas aeruginosa
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Strain DNA was extracted using the QIAamp DNA Mini Kit (50) (QIAGEN) following the manufacturer’s instructions. DNA was eluted in 30 μl of elution buffer. The genes, exotoxin S (exoS), exotoxin U (exoU), exotoxinA (exoA) were amplified using the specific primers as described earlier [41 (link), 42 (link)]. The PCR protocol involved initial denaturation step at 95°C for 10 min, followed by 40 cycles of 94°C for 2 min, annealing (30s at 57 to 65 °C) and 72°C for 1 min and the final extension step at 72°C for 5 min.
5
DNA Methylation Analysis of Tumor Samples
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DNA isolation was performed using a DNA isolation kit (Qiagen, QIAamp DNA Mini kit 50). The methylation status of promoter regions of P15, P16, RB1, and MGMT was determined by methylation-specific polymerase chain reaction. Therefore, 500 ng DNA of each tumor specimen as well as appropriate control samples were treated with bisulfite (Zymo Research, EZ DNA Methylation-Gold kit 200) (33 (link)). In summary, unmethylated cytosine was converted to uracil, whereas methylated cytosine remained unchanged. The modified DNA was recovered by ethanol precipitation and suspended in polymerase chain reaction (PCR) grade water. For analyzing the methylation status, the primer sequences listed in Table I were used (34 (link)–36 (link)). PCR was performed using a 25-µl reaction volume and 38 PCR cycles. All PCR products were electrophoretically separated on a 2% agarose gel. As a positive control, a chemically globally methylated DNA was used (Zymo Research, bisulfite-converted Human DNA). Genomic DNA isolated from a non-neoplastic dura mater tissue served as a negative control. In addition, each PCR included a control without any DNA template. An example of PCR results is presented in Fig. 1 .
6
Genetic Variation Analysis in Osteogenesis Imperfecta
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Genomic DNA of the probands, their parents and ethnically matched control was extracted from peripheral leukocytes with a QIAamp DNA Mini Kit (50) (Qiagen, Germany). All exons of FKBP10 and PLOD2, exon–intron junctions were amplified by polymerase chain reaction (PCR) in 23 reactions. Primers were designed using the software Oligo 7.0 (Table S1 ). Taq DNA polymerase (Biomed, China) and its standard buffer were used in all reactions under the following conditions: initial denaturation at 95°C for 2 min, followed by 35 cycles at 95°C for 30 s, 53–63°C for 30 s, and 72°C for 45/90 s. Direct sequencing reactions of PCR products were performed using BigDye Terminators Cycle Sequencing Ready Reaction Kit, version 3.1 (Applied Biosystems), and analyzed with an ABI 3130 automatic sequencer (Applied Biosystems) using standard methods. The results of sequencing were compared with the reference nucleotide sequence of FKBP10 (NM_021939.3) and PLOD2 (NM_182943.2). Genetic mutations identified in our patients were submitted to the OI database (https://oi.gene.le.ac.uk ).
7
Molecular Detection of Antibiotic Resistance
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DNA was extracted using the QIAamp DNA Mini Kit 50 (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. Molecular detection of each resistance gene for the different antibiotic groups was performed by polymerase chain reaction (PCR); the primers used are given in Supplementary Table S2 . The PCR reaction mixture (a total of 25 μL) contained 2 μL of extracted DNA as a template, 1 μL of forward primer, 1 μL of reverse primer, 4 μL of 5× hot firepol® blend master mix ready to load (Solis BioDyne, Tartu, Estonia), and 17 μL nuclease-free water. For the detection of each group of antibiotics, a different PCR cycle was performed, as shown in Table 2 . Agarose gel electrophoresis was applied to observe the bands of DNA.
8
Targeted SPOP Mutation Screening in Prostate Cancer
9
Extracting DNA and RNA from Frozen Tissue
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DNA and RNA were extracted from frozen tissue using the QIAamp DNA Mini Kit 50 and RNeasy Mini kit (Qiagen GmbH) according to the manufacturer's instructions.
10
Robust DNA Extraction and Quality Assurance
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Blood samples were collected using a 21-gage syringe to draw up to a volume of 5 ml in a vacutainer containing Tris-EDTA. For saliva samples, the participants were provided with collection tubes and were asked to provide a 2.5 ml sample. The participants were instructed to spit into the tube up to the red fill line marked on the tube (approximately 2.5 ml), excluding the froth. DNA was extracted using QIAamp DNA Mini Kit 50 (Cat# 51304, Qiagen). The DNA samples were was subjected to QIAXPERT for quantifying the amount of DNA and the purity was checked by measuring the 260/280 nm ratio. DNA samples were also subjected to agarose gel electrophoresis and, after passing through DNA quality check (Supplementary Table 1 ), were proceeded for Library Protocol.
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