Genomic DNA was isolated from HEK293T cells using a DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions. The mixture was treated with RNase A (Qiagen) to remove the residual RNA, after which the DNA was purified again with a DNeasy Blood & Tissue Kit (Qiagen). The in vitro transcribed crRNA (900 nM) was incubated with the purified dLbCpf1-BE protein (300 nM) at room temperature for 10 min. A total of 10 μg of purified genomic DNA was incubated with pre-complexed dLbCpf1-BE RNPs in a reaction volume of 400 μL in reaction buffer (50 mM Tris-HCl (Sigma-Aldrich) (pH 8.0), 25 mM KCl (Sigma-Aldrich), 2.5 mM MgSO4 (Sigma-Aldrich), 0.1 mM EDTA (Sigma-Aldrich), 10 % glycerol, 2 mM DTT (GoldBio), 10 μM ZnCl2 (Sigma-Aldrich)) at 37 °C for 8 h. The digested DNA was incubated with RNase A (50 μg/mL, Qiagen) to remove crRNA and then purified with a DNeasy Blood & Tissue Kit (Qiagen). Two microgram of purified DNA was incubated with USER (10 units, New England Biolabs) in a reaction volume of 200 μL at 37 °C for 2 h, and purified again with a DNeasy Blood & Tissue Kit (Qiagen). The target site was amplified by PCR and subjected to Sanger sequencing to check for dLbCpf1-BE-mediated deamination and USER-mediated formation of DNA SSBs.
Dneasy blood tissue kit
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom, Netherlands, Spain, Japan, China, Canada, France, Australia, Switzerland, Italy, Belgium, Denmark, Sweden
The DNeasy Blood & Tissue Kit is a DNA extraction and purification kit designed for the efficient isolation of high-quality genomic DNA from a variety of sample types, including whole blood, tissue, and cultured cells. The kit utilizes a silica-based membrane technology to capture and purify DNA, providing a reliable and consistent method for DNA extraction.
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5 685 protocols using dneasy blood tissue kit
1
dLbCpf1-BE-Mediated Genomic DNA Editing
2
Genome-Wide ABE Editing Analysis
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Genomic DNA was extracted from HT1-mCdHs using a DNeasy Blood & Tissue Kit (Qiagen). 8 μg of the genomic DNA was incubated with 32 μg of ABE pre-incubated with 24 μg of in vitro transcribed sgRNA at room temperature for 5 min, after which 300 μL of 2X BF buffer (Biosesang) was added and the reaction volume brought to 600 μL. That mixture was incubated at 37°C for 16 h. After RNase A (50 μg/mL, Thermo Scientific) treatment at 37°C for 15 min, the ABE-treated genomic DNA was purified using a DNeasy Blood & Tissue Kit (Qiagen). 3 μg of the purified DNA was digested with 8 units of Endo V (New England Biolabs) in a 200 μL reaction at 37 °C for 2 h. The genomic DNA was then purified using a DNeasy Blood & Tissue Kit (Qiagen). Whole genome sequencing was performed with 1 μg of the digested DNA using a HiSeq X Ten Sequencer (Illumina) at Macrogen (South Korea).
3
DNA Extraction from Insect Specimens
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Based on the defined criteria (see above) selected species were subjected to subsequent qPCR analysis. DNA from single individuals was extracted using the DNeasy® 96 Blood & Tissue Kit (Qiagen, Hilden, Germany). Unless otherwise noted, all buffers, racks, and tubes mentioned below are part of this extraction kit. The extraction procedure was a combination of the QIAGEN Supplementary Protocol DY14 (“Purification of total DNA from insects using the DNeasy® Blood & Tissue Kit”; August 2006) adapted to the DNeasy® 96 Blood & Tissue Kit as follows: For DNA extraction, 180 µL phosphate buffered saline (PBS, pH 7.2; 50 mM potassium phosphate, 150 mM NaCl; not part of the DNeasy® Blood & Tissue Kit) and a 3-mm diameter tungsten bead (Qiagen, Hilden, Germany; not part of the DNeasy® Blood & Tissue Kit) were added to every collection microtube containing an insect specimen. The samples were disrupted for 3 min at 30 Hz in the Tissue Lyser II (Qiagen, Hilden, Germany), and the lysates were collected by a short spin centrifugation of the 96-well microcentrifuge plate. Then 20 µL Proteinase K and 200 µL buffer AL (without ethanol) were added, vigorously mixed, and incubated for 10 min at 56 °C, and 200 µL 96% (v/v) ethanol (not part of the DNeasy® Blood & Tissue Kit) was added to each sample and processed following the instruction of the manufacturer.
4
Genomic and Circulating DNA Extraction
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Genomic DNA of tissue samples was extracted by using the QIAamp DNA FFPE Tissue Kit or the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). For cell-free DNA (cfDNA) extraction, plasma was separated by centrifugation at 1600×g for 10 min, then transferred to a new microcentrifuge tube and centrifuged at 16,000×g for another 10 min to remove any remaining cell debris. cfDNA was isolated from the plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Peripheral blood lymphocytes (PBLs) were used to extract germline genomic DNA from each patient with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). A Qubit fluorometer and the Qubit dsDNA HS (High Sensitivity) Assay Kit (Invitrogen, Carlsbad, CA USA) were used for DNA concentration measurement. And the size distribution of cfDNA was assessed with an Agilent 2100 BioAnalyzer and the DNA HS kit (Agilent Technologies, Santa Clara, CA, USA).
5
Selection and Characterization of Bacterial Mutants
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After 1 h of recovery from electroporation, we selected mutants as follows: Agar plates: We spread one portion of the culture on LB agar plates containing 40 µg/mL kanamycin (each plate received an appropriate volume of culture to generate 1000–2000 CFUs), and incubated at 37 °C overnight. On the next day, we first measured CFUs using a ProtoCOL3 automatic colony counter (Synbiosis), and then scraping off colonies from agar plates using a sterile cell spreader and 1 mL of LB. We recovered an equal number of mutants for each strain under investigation. We extracted DNA from a dilute sample of the pooled culture using the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer's instructions. Liquid culture: We transferred the equal volume of the culture directly into an Erlenmyer flask with fresh LB medium containing 40 µg/mL kanamycin (the volume of LB-kanamycin medium was 100X of the transferred culture volume). We shake-incubated the flask (175 r.p.m.) at 37 °C, and collected 1 mL sample when the OD600 of the culture reached 0.4, 0.8, and 1.2, which corresponds to early-, mid-, and late-log growth phase, respectively. We extracted DNA from each sample using the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer's instructions.
6
Specimen Collection and DNA Extraction
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Specimens were collected by a variety of methods ranging from collection by hand, to dredging, and deployment and recovery of wooden substrates (table 1 ). Specimens were frozen at −80 °C or preserved in 80–100% nondenatured ethanol following collection unless specified otherwise. For Xylophagaidae, siphon tissue was dissected from each bivalve, and total genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocols. Tissue samples collected in Alabama and Florida were preserved in 0.25 M EDTA, pH 8.0 (Sharpe et al. 2020 (link)). Total genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) and concentrated using the DNA Clean & Concentrator-25 Kit (Zymo Research) following manufacturer’s recommended protocols.
7
Extraction and Isolation of Queen and Sperm DNA
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Genomic DNA from queens was extracted from the hind legs using the DNeasy Blood & Tissue Kit (QIAGEN). Sperm present in the spermathecae of queens was collected and processed as described previously23 (link). Spermathecae were dissected in 1 × phosphate-buffered saline solution, and sperm clumps were separated from the membranes using insect pins. Genomic DNA from the male that mated with a queen was extracted from these sperm clumps using the DNeasy Blood & Tissue Kit (QIAGEN).
8
DNA Extraction and PCR Detection of Hgap Gene
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Total DNA was extracted from 100 µL whole blood using the DNeasy Blood & Tissue Kit (Qiagen, Germany), following the manufacturer’s protocol. DNA was extracted from 25 mg liver tissue, after the tissue was homogenized using a rotor-stator homogenizer and probe (Qiagen). The total DNA from liver tissue was extracted using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol.
PCR primers used for detecting the Hgap gene to confirm the presence of DCH were synthesized (forward: 5’-GTGCTCTGATAACCGTATGCTC-3’; reverse: 5’-CTAGAATGGCTACATGGGGTTAG-3’) as reported previously [1 ]. Conventional PCR was performed using the Bioline MyTaq hot-start polymerase (Bioline, Australia) according to the manufacturer’s protocol: 300 ng DNA template, 5 × MyTaq buffer, 20 µM final primer concentration in a total 25 µL reaction. The PCR cycling conditions were as follows: Initial denaturation at 95℃ for 1 min, followed by 40 cycles of denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 10 s and final extension at 72℃ for 5 min. The PCR products from both the blood and liver samples were analysed with (1.5%) agarose gel electrophoresis, from which specific bands containing the PCR products were purified, excised and verified. The amplicons were verified using the Sanger DNA sequencing method.
PCR primers used for detecting the Hgap gene to confirm the presence of DCH were synthesized (forward: 5’-GTGCTCTGATAACCGTATGCTC-3’; reverse: 5’-CTAGAATGGCTACATGGGGTTAG-3’) as reported previously [1 ]. Conventional PCR was performed using the Bioline MyTaq hot-start polymerase (Bioline, Australia) according to the manufacturer’s protocol: 300 ng DNA template, 5 × MyTaq buffer, 20 µM final primer concentration in a total 25 µL reaction. The PCR cycling conditions were as follows: Initial denaturation at 95℃ for 1 min, followed by 40 cycles of denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 10 s and final extension at 72℃ for 5 min. The PCR products from both the blood and liver samples were analysed with (1.5%) agarose gel electrophoresis, from which specific bands containing the PCR products were purified, excised and verified. The amplicons were verified using the Sanger DNA sequencing method.
9
Tick DNA Extraction for Borrelia
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All ticks were washed with 75% ethanol and rinsed thrice with sterile water to remove environmental contaminants. Except for the 182 I. persulcatus samples used for the isolation and culture of B. burgdorferi, the remaining 100 I. persulcatus and 13 D. silvarum samples were used for DNA extraction. Individual tick samples were homogenized in 180 μL buffer ATL, then genomic DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Similarly, the genomic DNA of human blood samples was extracted from anticoagulated blood using the DNeasy Blood & Tissue Kit (QIAGEN, Germany). Each time DNA extraction was performed, an extraction control (water) was added. All DNA samples from both ticks and human blood were stored at −80°C until use.
10
Tumor and Cell-free DNA Extraction
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Tumor DNA was extracted from fresh frozen tissue using the DNeasy Blood & Tissue Kit® (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Plasma was separated from whole blood by centrifugation for 10 min at 3000 rpm (1840×g) and stored at −80 °C until further use. The samples were centrifuged again for 10 min at 13,300 rpm (16,000×g) prior to DNA extraction to remove debris. Cell-free DNA (cfDNA) was isolated from 1 mL of plasma using the QIAamp Circulating Nucleic Acid Kit® (Qiagen) according to the manufacturer's instructions and eluted in 50 μL of AVE buffer (Qiagen). PBL were separated from whole blood by centrifugation for 10 min at 3000 rpm (1840×g) twice and stored at −80 °C until further use in the pellet state. DNA was extracted from the PBL pellet using the DNeasy Blood & Tissue Kit® (Qiagen) according to the manufacturer's instructions.
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