The bacterial DNA was extracted from colon and caecum digesta and purified using the QIAamp DNA Stool Mini Kit (No. 51504; Qiagen, West Sussex, UK) as described by Castillo et al. [37 (link)]. Briefly, each frozen digesta sample (0.3 ~ 0.5 g) was thawed and homogenized in the InhibitEX buffer (Qiagen, West Sussex, UK) and centrifuged to obtain the supernatant fluid. After the addition of proteinase K (Qiagen, West Sussex, UK) to the supernatant fluid, the solution was mixed by vortexing and then centrifuged to collect the supernatant fluid. The latter was mixed with ethanol (96–100%), and DNA was purified by using the QIAamp spin column (Qiagen, West Sussex, UK). Total DNA was quantified using the NanoDrop® ND-1000A UV-VIS spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at an OD of 260 nm, and its purity was assessed by determining the OD260nm/OD280nm ratio. All of the samples had an OD260nm/OD280nm ratio of 1.7 to 1.9. The length of the genomic DNA in each sample was determined using 1% denatured agarose gel electrophoresis. The microbial DNA was stored at −20 °C until qPCR analysis.
Qiaamp spin column
Manufactured by Qiagen
Sourced in United Kingdom, Germany
The QIAamp spin column is a laboratory centrifugation device used for the extraction and purification of nucleic acids, such as DNA and RNA, from various biological samples. It utilizes a silica-based membrane to selectively bind nucleic acids, allowing for their isolation and separation from other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna stool mini kit, by Qiagen (4 mentions)
Nanodrop nd 1000a uv vis spectrophotometer, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Cfx software, by Bio-Rad (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Inhibitex buffer, by Qiagen (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Rw1 buffer, by Qiagen (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Asl buffer, by Qiagen (1 mentions)
Zirconium beads, by Qiagen (1 mentions)
10 protocols using qiaamp spin column
1
Extracting Bacterial DNA from Colon and Caecum
2
Microbial DNA Extraction from Stool
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The bacterial DNA was extracted from the intestinal content using the QIAampDNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The extracted DNA was purified by using the QIAamp spin column (Qiagen, Hilden, Germany). Total DNA was quantified by determining the OD 260 nm/OD 280 nm ratio using the NanoDrop®ND-1000A UV-VIS spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The quality of microbial DNA in each sample was evaluated by 1% denatured agarose gel electrophoresis and the quantity of DNA with Picogreen assay. The microbial DNA were normalized with 10 mM Tris buffer (pH 8.5) to 5 ng/ul.
3
Quantitative PCR RNA Extraction and Synthesis
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Cells used for qPCR analysis were lysed in RLT buffer (Qiagen, Sollentula, Sweden) and mixed with 70% ethanol. Cell samples were then transferred to a QIAamp spin column (Qiagen) centrifuged and washed (RW1 buffer (Qiagen)), then treated with DNase (15 min, room temperature), washed and centrifuged. Samples were resuspended in H2O and the RNA concentration was measured by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized with the High-capacity cDNA reverse transcription kit (Thermo Fisher Scientific) according to the supplier’s recommendations. For the generation of cDNA, 0.1 µg of RNA was used in a total reaction volume of 10 µL using random hexamer priming.
4
Microbial Diversity Analysis via 16S rRNA Sequencing
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Total DNA was isolated from the contents of cecum using a QIAamp DNA stool mini kit (Qiagen, Hilden, Germany). The precipitated DNA was washed and purified using a QIAamp spin column (Qiagen) according to the manufacturer's instructions and finally eluted in 200 µL of purified water.
The microbial composition of these samples was determined by sequencing the 16S rRNA V6, V7, V8, and partial V9 variable regions (434 bp). The V6, V7, V8, and partial V9 regions of the 16S rRNA gene were amplified using the universal primers, 5′-AACGCGAAGAACCTTAC-3′ and 5′-CGGTGTGTACAAGACCC-3′.
The microbial composition of these samples was determined by sequencing the 16S rRNA V6, V7, V8, and partial V9 variable regions (434 bp). The V6, V7, V8, and partial V9 regions of the 16S rRNA gene were amplified using the universal primers, 5′-AACGCGAAGAACCTTAC-3′ and 5′-CGGTGTGTACAAGACCC-3′.
5
Genomic DNA Extraction from Biological Samples
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DNA extraction was done by QIAamp DNA Mini Kit according to manufacturer's recommendations. The pellet was liquefied by adding 200 μl of PBS solution, followed by gentle vortexing. A volume of 200 μl of sample was added to 20 μl of QIAGEN Protease (Proteinase K) in a 1.5 ml microcentrifuge tube. Next, 200 μl of Buffer AL was added to the mixture and mixed thoroughly. The mixture was then incubated at 56°C for 10 min and centrifuged briefly to remove the drops from inside. The mixture was then loaded to QIAamp spin column (Qiagen) and was centrifuged at 8000 rpm for 1 min. The filtrate obtained in 2 ml collecting tube was discarded. Then, 500 μl of buffer AW1 was added to QIAamp spin column and was centrifuged at same rpm for 1 min. The filtrate obtained in the tube was discarded. The wash procedure was repeated by adding 500 μl of buffer AW2 to the spin column and centrifuging at 14000 rpm for 3 min. The filtrate obtained in the tube was again discarded. Finally, 200 μl of buffer AE was added to the spin column and was centrifuged at 8000 rpm for 1 min. The elute containing the pure genomic DNA was stored at -20°C.
6
Schistosoma Detection by qPCR in CVL and Swabs
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Detection of the Schistosoma-specific internal-transcribed-spacer-2 (ITS2) target by qPCR was performed at LUMC, as previously described (Supplementary Data ) [18 (link), 29 (link)]. DNA extraction of 200 µL of CVL or cervical or vaginal swab fluid was done with QIAamp spin columns (QIAGEN Benelux, Venlo, the Netherlands) according to manufacturer’s guidelines. The qPCR output was reported in cycle threshold values (Ct values), and parasite DNA loads were categorized by the following prespecified values: high (Ct < 30), moderate (30 ≤ Ct < 35), low (35 ≤ Ct < 50), and negative (no amplification) [30 (link)].
7
Schistosoma DNA Extraction and Detection
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DNA extraction and PCR set up was performed at LUMC, using a custom automated liquid handling station (Hamilton, Switzerland), as previously described (23 (link)). DNA was extracted from 200 µl of specimen (cervical swab, vaginal swab, CVL): with QIAamp spin columns (QIAGEN Benelux; Venlo, The Netherlands). Detection of the schistosome-specific internal-transcribed-spacer-2 (ITS2) target was performed by real-time PCR as previously described (23 (link), 29 (link)). This PCR does not differentiate between Schistosoma species. DNA amplification and detection were performed with the CFX96 Real Time PCR Detection System (BioRad, California, USA). The output in cycle quantification value (Cq), reflecting the parasite-specific DNA load in the tested sample, was analyzed using BioRad CFX software. Parasite DNA loads were categorized by the following pre-specified Cq thresholds: high (Cq<30), moderate (30≤ Cq <35), low (35≤ Cq <50) and negative (no Cq detected), as previously described (30 (link)).
8
Schistosoma ITS-2 qPCR Detection Protocol
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Detection of the Schistosoma-specific internal-transcribed-spacer-2 (ITS-2) target by qPCR was performed at Leiden University Medical Center, as previously described [9 (link), 22 (link)]. Deoxyribonucleic acid extraction of 200 µL CVL, cervical, or vaginal swab fluid was done with QIAamp spin columns (QIAGEN Benelux, Venlo, The Netherlands) according to manufacturer's guidelines. The qPCR output was reported in cycle threshold (Ct) values, and parasite DNA loads were categorized by the following prespecified values: high (Ct < 30), moderate (30 ≤ Ct < 35), low (35 ≤ Ct < 50), and negative (no amplification) [23 (link)].
9
Schistosoma ITS2 Real-Time PCR
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DNA extraction and PCR was performed at LUMC as previously described, using a custom automated liquid handling station (Hamilton, Switzerland) [20 (link), 31 (link)]. DNA was extracted from 200 μL of specimen (cervical swab, vaginal swab, CVL) with QIAamp spin columns (QIAGEN Benelux, Venlo, the Netherlands). Detection of the schistosome-specific internal-transcribed-spacer-2 (ITS2) target was performed by real-time PCR as previously described [18 (link), 31 (link)]. This PCR does not differentiate between Schistosoma species. DNA amplification and detection were performed with the CFX96 Real Time PCR Detection System (BioRad, Hercules, CA, USA). The output in threshold cycles (Cts), reflecting the parasite-specific DNA load in the tested sample, was analyzed using BioRad CFX software. Parasite DNA loads were categorized by the following prespecified Ct thresholds: high (Ct < 30), moderate (30 ≤ Ct < 35), low (35 ≤ Ct < 50), and negative (no Ct detected), as previously described [32 (link)].
10
Fecal DNA Extraction from HIV Patients
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Total DNA was extracted from the 13 frozen fecal samples of HIV-infected patients using a modification of the Qiagen stool procedure and the QIAamp® DNA Stool Mini Kit (Qiagen, Courtaboeuf, France) [5 (link)]. Briefly, aliquots of 200 mg of feces were added to tubes containing a 200 mg mixture of 0.1, 0.5, and 2 mm zirconium beads and 1.5 ml of ASL buffer (Qiagen). The sample was bead-beaten at 3200 rpm for 90 seconds, followed by heating at 95°C for 10 minutes. The final pellet was suspended in 180 μl of tissue lysis buffer and incubated with proteinase K for 2 hours at 55°C. Then, DNA was prepared from the solution by using QIAamp spin columns (Qiagen) in an Eppendorf microcentrifuge, following the manufacturer’s instructions.
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