The collected samples, including the roots, feces, sediments and negative controls prepared in the field (PBS collected and manipulated exactly as the field samples), were subjected to molecular identification. After the manual lysis of root plants in the homemade sterile Trans MU transport medium using sterile disposable pestles, 500 μL of each sample was transferred to a 1.5 ml Eppendorf tube. A volume of 200 μL of lysis buffer G2, 20 μL of proteinase K (Qiagen GmbH, Hilden, Germany) and a small amount of glass powder was added. The samples underwent three runs of fast prep (6.5 m/s) followed by incubation at 56°C for three hours and centrifugation for one minute at 1,100 g. A 200-μL volume of supernatant was transferred into a new tube, and total DNA extraction was performed on an EZ1 machine (Qiagen). The extracted DNA samples were stored at -20°C until use.
Ez1 machine
Manufactured by Qiagen
The EZ1 machine is a compact and automated nucleic acid extraction system developed by Qiagen. It is designed to efficiently purify DNA, RNA, or viral nucleic acids from a variety of sample types. The EZ1 machine utilizes magnetic-particle technology to streamline the extraction process, providing consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Ez1 dna tissue kit, by Qiagen (3 mentions)
Ez1 advanced xl machine, by Qiagen (2 mentions)
Proteinase k solution, by Qiagen (2 mentions)
Genomic dna screentape, by Agilent Technologies (2 mentions)
Proteinase k, by Qiagen (1 mentions)
Transilluminator, by Bio-Rad (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Labtype sso hd kits, by Thermo Fisher Scientific (1 mentions)
5 protocols using ez1 machine
1
Molecular Identification of Environmental Samples
2
Evaluating DNA Degradation in Tissue Samples
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Upon receipt of the tissue from the NeuroBioBank, DNA degradation was evaluated by isolating DNA from the tissue using the Qiagen EZ1 Advanced XL machine (Qiagen Catalog No. 9001875) and EZ1 DNA Tissue Kit (Qiagen Catalog No. 953034). Briefly, tissue was dissected with a scalpel on a cold block to produce approximately 34 mg of tissue. Then, 190μl of G2 buffer (Qiagen Catalog No. 1014636) and 10μl Proteinase K solution (Qiagen Material No. 101406) was added to the tissue. These samples were then vortexed for 15 seconds, briefly centrifuged for 15 seconds at maximum speed on the benchtop centrifuge and placed onto the thermo-mixer at 56°C until the tissue was dissolved (approximately 2 hours). Subsequently, all samples were pipette mixed, briefly centrifuged for 15 seconds at maximum speed on the benchtop centrifuge, and loaded onto the Qiagen EZ1 machine for extraction, with use of the EZ1 DNA Reagent Cartridge for tissue (from Qiagen Catalog No. 953034). The elution value was set as 200 μl for each of the extraction methods, and once extracted, all samples were stored at −20°C until quantification was carried out. Genomic DNA Screen Tape (Agilent Catalog No. 5067–5365) was used to determine DNA length. Tissues with fragmented DNA were not selected for further studies.
3
Acinetobacter baumannii DNA Binding Assay
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A. baumannii (ATCC BAA‐1709) was cultured in MHB medium overnight (100 rpm, 37 °C). The bacterial suspension (50 mL) was then centrifuged and the pellet was lysed by G2 buffer. The genomic DNA was extracted using an EZ1 DNA Tissue Kit (48) in the EZ1 machine (QIAGEN) following the EZ1 DNA Tissue protocols. The gene binding ability of pEt_20 was investigated by agarose gel electrophoresis. DNA extracted from A. baumannii BAA‐1709 was mixed with pEt_20 at various polymer to DNA mass ratios (1–20). Briefly, 9.5 µL of the pEt_20/gene complex solution containing 300 ng of gene and corresponding polymer at their respective mass ratios was mixed with 0.5 µL of 5× DNA loading dye. The mixture (7 µL) was loaded into individual wells of 1% agarose gel containing SYBR Safe DNA Gel Stain (Thermofisher Scientific, USA) at a ratio of 1:10 000 1× TAE buffer. The same amount of naked DNA was used as the control. The gel was run at 120 mV for 20 min in 1× TAE buffer. Following completion of the assay, the gel was imaged using a gel imaging system fitted with a transilluminator (Bio‐rad, U.S.A.).
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A. baumannii (ATCC BAA‐1709) was cultured in MHB medium overnight (100 rpm, 37 °C). The bacterial suspension (50 mL) was then centrifuged and the pellet was lysed by G2 buffer. The genomic DNA was extracted using an EZ1 DNA Tissue Kit (48) in the EZ1 machine (QIAGEN) following the EZ1 DNA Tissue protocols. The gene binding ability of pEt_20 was investigated by agarose gel electrophoresis. DNA extracted from A. baumannii BAA‐1709 was mixed with pEt_20 at various polymer to DNA mass ratios (1–20). Briefly, 9.5 µL of the pEt_20/gene complex solution containing 300 ng of gene and corresponding polymer at their respective mass ratios was mixed with 0.5 µL of 5× DNA loading dye. The mixture (7 µL) was loaded into individual wells of 1% agarose gel containing SYBR Safe DNA Gel Stain (Thermofisher Scientific, USA) at a ratio of 1:10 000 1× TAE buffer. The same amount of naked DNA was used as the control. The gel was run at 120 mV for 20 min in 1× TAE buffer. Following completion of the assay, the gel was imaged using a gel imaging system fitted with a transilluminator (Bio‐rad, U.S.A.).
4
High-Resolution HLA Genotyping by Luminex
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Study samples were HLA genotyped by Luminex-based sequence-specific oligonucleotide typing assays (Supplemental Table 5 ). Genomic DNA was extracted from cells using the Qiagen EZ-1 machine. Intermediate resolution of all HLA loci (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1) were performed using the LABType SSO HD kits (One Lambda). Briefly, the DNA was amplified using primers that were specific for each of the 11 HLA loci. The PCR product was biotinylated and denatured to make single-stranded DNA. This DNA was then hybridized to a series of probes specific for nucleotide sequences that were used to define the HLA genes. Each probe was bound to a uniquely fluorescent coded Luminex bead. The mixture was then washed to remove any excess, unbound PCR product. The bound PCR product was labeled with streptavidin conjugated with a PE fluorescent tag (SAPE). The DNA-bead complexes were run on a Luminex 100 platform and analyzed using HLA-FUSION v3.5.6 software.
5
Evaluating DNA Degradation in Tissue
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