From swab tube, 140 μl sample was transferred to 1.5 mL screw-cap microcentrifuge tube and treated with 560 μl AVL, containing carrier RNA, followed by 560 μl molecular-grade 100% ethanol (Fisher Scientific). Samples were then taken out of the Class I MSC and CL-3 lab as AVL is known to inactivate SARS-CoV-2, transferred into QIAamp mini spin columns (Qiagen) and centrifuged according to manufacturer’s instructions. Two wash steps were performed, with 714 μl buffer AW1 and 714 μl buffer AW2 (both Qiagen). RNA was then eluted from the columns with 40 μl RNase-free water (Ambion), followed by a second 40 μl elution to maximise RNA yield and giving a final RNA sample volume of 80 μl.
Buffer aw1
Manufactured by Qiagen
Sourced in Germany
Buffer AW1 is a lab equipment product designed to facilitate DNA purification and extraction processes. It serves as a fundamental component in various molecular biology and genomic applications. The core function of Buffer AW1 is to provide the necessary buffer conditions for the effective binding of DNA to specialized purification columns or matrices.
Automatically generated - may contain errors
Lab products found in correlation
Buffer aw2, by Qiagen (7 mentions)
Ae buffer, by Qiagen (5 mentions)
Buffer al, by Qiagen (5 mentions)
Atl buffer, by Qiagen (4 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Qiaamp mini spin column, by Qiagen (2 mentions)
Molecular grade 100 ethanol, by Thermo Fisher Scientific (2 mentions)
714 μl buffer aw2, by Qiagen (2 mentions)
Rnase free water, by Thermo Fisher Scientific (2 mentions)
Ave buffer, by Qiagen (2 mentions)
Proteinase k, by Qiagen (2 mentions)
Qiaampminielute spin column, by Qiagen (2 mentions)
Qiaamp dna micro kit, by Qiagen (2 mentions)
Ethanol, by Carl Roth (1 mentions)
Dneasy blood and tissue handbook, by Qiagen (1 mentions)
Biosprint 96 one for all vet kit, by Qiagen (1 mentions)
Magmax express 96, by Thermo Fisher Scientific (1 mentions)
Magattract suspension g, by Qiagen (1 mentions)
96 well plate, by Qiagen (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
13 protocols using buffer aw1
1
SARS-CoV-2 RNA Extraction from Swabs
2
Automated RNA Extraction using KingFisher
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The KingFisher™ (KF) Duo system was applied with a 12 pin magnet head which enabled processing of 12 samples per run using microtiter 96 deepwell plates (Thermo Fisher). Prior to the extraction process, the deepwell plates were filled with the following reagents: lysis buffer VXL, MagAttract Suspension B, buffer AW1, buffer AW2 (Qiagen), 100% Ethanol (Carl Roth GmbH, Karlsruhe, Germany), nuclease-free water and buffer AVE (Qiagen) [see Additional file 1 : Table S1]. For extraction, 100 μl of the sample were added to the lysis buffer and mixed by pipetting. After that, binding buffer ACB was added and automated extraction was executed and completed within 8 min using the protocol indicated in Table 1 . The extracted RNA was eluted in 100 μl and subsequently transferred to 1.5 ml tubes for storage.
3
DNA Extraction from Blood and Tissue
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DNA was extracted according to the DNeasy Blood and Tissue Handbook (July 2006, Qiagen), with certain modifications designed to enhance recovery. Briefly, a 150-μL aliquot of the homogenate or 50-μL aliquot of blood was mixed with 70 μL of ATL buffer and proteinase K (9:1, v/v), whereas calibration standards were mixed with 70 μL of the working cell lysate prepared as described above. For blood samples, an additional 100 μL of buffer ATL and proteinase K (9:1, v/v) was added in order to obtain equal volumes of blood and tissue homogenate samples. To prepare the blood samples, 205 μL of buffer AL (Qiagen) was added, followed by incubation at 56 °C for 30 min, after which 205 μL of ethanol was added. For tissue samples, 410 μL of buffer AL and ethanol (1:1, v/v) was added, followed by incubation at 56 °C for 15 min, and the samples were subsequently loaded onto a DNeasy 96 plate. The plate was washed twice with 500 μL of buffer AW1 (Qiagen) and then washed with 500 μL of buffer AW2 (Qiagen). Finally, DNA was eluted from each sample using 200 μL of buffer AE (Qiagen).
4
Horse SMLN DNA Extraction Protocol
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DNA extraction was performed under a biosafety cabinet with sterile labware to reduce environmental contamination and DNA carryover between samples. DNA was extracted from all samples using a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). Based on the manufacturer’s recommendations, a 25 mg piece of SMLN from each horse was placed in individual Petri dishes and minced. The minced tissue was transferred to a clean microcentrifuge tube containing Buffer ATL (Qiagen, Valencia, CA, USA) and 40 µL proteinase K, then digested overnight at 56 °C. A mixture of the tissue lysate, buffer AL and ethanol was pipetted into a DNeasy Mini spin column (Qiagen), then centrifuged for 1 min at 13,300 rpm. The supernatant was discarded, and Buffer AW1 (Qiagen) was added to the spin column, and then centrifuged again. The spin column was then washed with Buffer AW2 (Qiagen), which was centrifuged for 3 min. The DNA was eluted in 100 µL of Buffer AE (Qiagen, Valencia, CA, USA) via incubation at room temperature for 1 min before centrifugation for 1 min at 13,300 rpm. The DNA was stored at −20 °C until downstream processing took place.
5
SARS-CoV-2 RNA Extraction from Swab Samples
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From swab tube, 140 μl sample was transferred to 1.5 mL screw-cap microcentrifuge tube and treated with 560 μl AVE, containing carrier RNA, followed by 560 μl molecular-grade 100% ethanol (Fisher Scientific). Samples were then taken out of the Class I MSC and CL-3 lab as AVL is known to inactivate SARS-CoV-2, transferred into QiaAmp mini spin columns (Qiagen) and centrifuged according to manufacturer’s instructions. Two wash steps were performed, with 714 μl buffer AW1 and 714 μl buffer AW2 (both Qiagen). RNA was then eluted from the columns with 40 μl RNase-free water (Ambion), followed by a second 40 μl elution to maximise RNA yield and giving a final RNA sample volume of 80 μl.
6
DNA Purification Using Biosprint® 96 Kit
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DNA was purified using the Biosprint® 96 One-for-all Vet kit (Qiagen). Briefly, 400 µL of the supernatant obtained above was transferred to a 96-well lysate plate containing 40 µL proteinase K (Qiagen) to which 300 µL of magnetic bead suspension comprising 25 µL MagAttract suspension G (Qiagen) in 300 µL isopropanol (Sigma-Aldrich) was added. Magnetic bead-based purification of DNA was performed using an automated platform (MagMAX Express-96; Life Technologies), which included transfer of the bead-bound DNA through three wash steps using two different wash buffers (Buffer AW1 and RPE, Qiagen) contained in separate 96-well plates (Qiagen), air drying of any wash buffer residue and DNA elution in Buffer AVE (Qiagen). The eluted DNA was stored at −80°C before use. The DNA extract was tested neat and also as a fivefold dilution in Buffer AVE (Qiagen).
7
Optimized Genomic DNA Extraction from Buffy Coat
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All blood samples were centrifuged sequentially at 2990 rpm for 10 min each; then, buffy coats (200 µL) were collected and stored at −80 °C as plasma. The pretreatment process for DNA extraction of sample is as follows: The 200 μL of buffy coat was treated with 20 μL proteinase K to remove erythrocyte and ascites. The reaction ended, buffy coat and ascites were mixed with 200 μL lysis buffer by pulse-vortexing for 15 s, then incubated at 57 °C for 15 min. The sample was added to 200 μL 95% ethyl alcohol (Samchun, Seoul, Korea) and mixed again by pulse-vortexing for 15 s. The bead was reacted with the pretreated sample for 10 min. The bead was carefully dipped into 350 μL buffer AW1 (Qiagen, Hilden, Germany) for 60 s, then dipped into 50 μL Rnase/Dnase free water for 3 min. The elution was stored at −80 °C. DNA was extracted using a QIAamp DNA Mini kit (Qiagen) following the instructions provided by the manufacturer as described previously [21 (link),22 (link)].
8
Automated DNA Extraction from Cells
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Genomic DNA was extracted from cell pellets using a modified version of the Qiagen DNeasy Blood and Tissue Kit protocol. First, pellets in 96DW plates were removed from −80 °C and thawed in a room temperature water bath. Each pellet was resuspended in 180 μL of enzymatic lysis buffer (20 mM Tris–HCl (Invitrogen), 2 mM sodium EDTA (Sigma-Aldrich), 1.2% Triton X-100 (Sigma-Aldrich), 20 mg/mL lysozyme from chicken egg white (Sigma-Aldrich)). Plates were then covered with a foil seal and incubated at 37 °C for 30 min with orbital shaking at 600 RPM. Then, 25 μL of 20 mg mL−1 Proteinase K (VWR) and 200 μL of Buffer AL (QIAGEN) were added to each sample before mixing with a pipette. Plates were then covered by a foil seal and incubated at 56 °C for 30 min with orbital shaking at 600 RPM. Next, 200 μL of 100% ethanol (Koptec) was added to each sample before mixing and samples were transferred to a nucleic acid binding (NAB) plate (Pall) on a vacuum manifold with a 96DW collection plate. Each well in the NAB plate was then washed once with 500 μL buffer AW1 (QIAGEN) and once with 500 μL of buffer AW2 (QIAGEN). Samples were then eluted into a clean 96DW plate from each well using 110 μL of buffer AE (QIAGEN) preheated to 56 °C. Genomic DNA samples were stored at −20 °C until further processing.
9
Plasma cfDNA Extraction and Analysis
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Blood plasma was collected from 3 to 5 mL human blood samples after blood aliquot was centrifuged at 2990 rpm for 10 min. The plasma was pre-treated with proteinase K in a 10:1 volume ratio (200 μL: 20 μL) for 3 h at 37 °C. The samples were mixed with 200 μL of lysis buffer and incubated for 10 min at 37 °C, followed by additional mixing with 200 μL of 95% ethyl alcohol. The PDA–silica-coated beads were then dipped into the pre-treated samples with 20 μL of CaCl2 solution. The DNA fragments were reacted with the PDA–silica hybrids for 10 min under gentle agitation. The beads were then washed with 350 µL of AW1 buffer (Qiagen, Hilden, Germany) and stored in 50 µL of RNase/DNase free water. The plasma cfDNA fragments were measured using the Agilent Bioanalyzer 2100 instrument and High Sensitivity DNA Kit, a microfluidics-based platform, to determine the mean size for each defined smear region of plasma cfDNA and display electropherogram for each sample. The concentration of cfDNA was determined to range between 100 and 500 bp (Supplementary Figure S1 ). The details can be found in our previous publications [29 (link),31 (link)].
10
Liposarcoma DNA Extraction Protocol
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Extraction of DNA from liposarcoma tumor tissues and cell lines was performed using the QIAamp DNA Micro kit (Qiagen, Valencia, CA). The extraction was carried out according to the manufacturer’s instructions. Briefly, liposarcoma tumor tissue samples (approximately 50 mg) or cell pellets from cultured cell lines (approximately 8 mg) were transferred to a 1.5 mL microcentrifuge tube and 180 μL of buffer ATL (Qiagen) was immediately added. After equilibrating to room temperature (25°C), 20 μL of proteinase K was added and mixed by vortexing for 15 seconds. The sample tube was incubated at 56°C overnight until the sample was completely lysed. The next day, 200 μL buffer AL (Qiagen) was added and mixed by vortexing for 15 seconds. Subsequently, 200 μL of ethanol (96%–100%) was added. The mixture obtained was loaded on a QIAampMiniElute spin column and washed with AW1 buffer, followed by AW2 buffer (Qiagen). DNA was eluted with 60 μL of bufferAE (Qiagen) and preserved at −20°C until use. Quantity and quality of the DNA was determined using a spectrophotometer (DU640, Beckman Instruments, Inc, Fullerton, CA) and agarose gel electrophoresis.
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