Primers and probes were purchased from IDT (Coralville, IA, USA) and from LGC, Biosearch Technologies (Petaluma, CA, USA). GUY1 and RPS6 linear fragments were purchased from GeneWiz (South Plainfield, NJ, USA). YG2 linear fragment and KLH07 plasmid were purchased from IDT (Coralville, IA, USA). EZ1 Advance XL and EZ1 DNA Tissue Kits were purchased from Qiagen (Hilden, Germany). TaqMan™ Fast Advanced Master Mix, 10 mM dNTP Mix, 25 mM MgCl2, and DEPC-Treated Water were purchased from ThermoFisher Scientific (Waltham, MA, USA). KAPA3G Plant PCR Kit was purchased from Roche Sequencing and Life Science (Indianapolis, IN, USA). ZR BashingBead (2 mm) Lysis Tubes was purchased from Zymo Research (Irvine, CA, USA). Mini-Beadbeater-16 was purchased from BioSpec Product, Inc. (Bartlesville, OK, USA). Proteinase K originated from the Qiagen (Hilden, Germany) EZ1 DNA Tissue kit.
Ez1 dna tissue kit
Manufactured by Qiagen
Sourced in Germany, France, United States, United Kingdom
The EZ1 DNA Tissue Kit is a lab equipment product from Qiagen. It is designed for automated extraction and purification of genomic DNA from a variety of tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Biorobot ez1, by Qiagen (30 mentions)
Proteinase k, by Qiagen (19 mentions)
Epitect bisulfite kit, by Qiagen (11 mentions)
Pyromark assay design software 2, by Qiagen (10 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Ez1 advanced xl, by Qiagen (9 mentions)
G2 lysis buffer, by Qiagen (7 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (7 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Biorobot ez1 system, by Qiagen (6 mentions)
Ez1 advanced xl instrument, by Qiagen (6 mentions)
Allprep dna rna mini kit, by Qiagen (6 mentions)
Buffer g2, by Qiagen (6 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (5 mentions)
Biorobot ez1 workstation, by Qiagen (5 mentions)
Atl buffer, by Qiagen (5 mentions)
Qubit assay, by Thermo Fisher Scientific (5 mentions)
Ez1 advanced xl robot, by Qiagen (4 mentions)
Miseq sequencer, by Illumina (4 mentions)
Pyromark q96 system, by Qiagen (4 mentions)
193 protocols using ez1 dna tissue kit
1
Quantitative PCR Protocol for GUY1, RPS6, and YG2
2
Genomic DNA Extraction from Tissue and Tachyzoites
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Genomic DNA was extracted from 300 μL of digested pellets of all collected tissue samples using an EZ1® DNA Tissue Kit (Qiagen, Germany), according to the manufacturer’s instructions for tissue samples.
Genomic DNA from a suspension of 2.105 cultured RH-strain tachyzoites was extracted using the same protocol (EZ1® DNA Tissue Kit, Qiagen, Germany). A standard curve was developed, with four 10-fold serial dilutions of the genomic DNA, corresponding to 1 × 100 to 1 × 103 tachyzoites/μL.
Genomic DNA from a suspension of 2.105 cultured RH-strain tachyzoites was extracted using the same protocol (EZ1® DNA Tissue Kit, Qiagen, Germany). A standard curve was developed, with four 10-fold serial dilutions of the genomic DNA, corresponding to 1 × 100 to 1 × 103 tachyzoites/μL.
3
Molecular Detection of Monkeypox and Orthopoxviruses
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Crust and vesicular swab specimens were processed for testing and storage at INRB.
Swab specimens were hydrated in 1.5 mL of phosphate-buffered saline (PBS) and then spun on a benchtop microcentrifuge for 30 seconds, after which the supernatant was eluted. Crust specimens were first homogenized in 0.5 mL of PBS solution, then an additional 1.0 mL of PBS was added, and the sample was spun on a benchtop centrifuge for 30 seconds to settle particulate matter. For each sample type, approximately 1.5 mL of supernatant was produced.
For each specimen, 100 μL of supernatant was used for the MPX/OPX GeneXpert assay and 100 μL was used for DNA extraction (Qiagen EZ1 DNA Tissue Kit, Germantown, MD) and real-time OPX PCR at INRB.23 (link) Additionally, 800 μL of the supernatant from each specimen was sent to the CDC for independent testing and validation, where 100 μL was used for a duplicate GeneXpert assay and 100 μL was used for DNA extraction and real-time MPX-generic PCR. At CDC, DNA was extracted from the supernatant using the EZ1 DNA Tissue Kit (Qiagen) in conjunction with the EZ1 platform (Qiagen).
All steps requiring specimen manipulation, including swab processing, crust homogenization, elution in PBS, and transfer to the GeneXpert cartridge were conducted within a certified biosafety cabinet.
Swab specimens were hydrated in 1.5 mL of phosphate-buffered saline (PBS) and then spun on a benchtop microcentrifuge for 30 seconds, after which the supernatant was eluted. Crust specimens were first homogenized in 0.5 mL of PBS solution, then an additional 1.0 mL of PBS was added, and the sample was spun on a benchtop centrifuge for 30 seconds to settle particulate matter. For each sample type, approximately 1.5 mL of supernatant was produced.
For each specimen, 100 μL of supernatant was used for the MPX/OPX GeneXpert assay and 100 μL was used for DNA extraction (Qiagen EZ1 DNA Tissue Kit, Germantown, MD) and real-time OPX PCR at INRB.23 (link) Additionally, 800 μL of the supernatant from each specimen was sent to the CDC for independent testing and validation, where 100 μL was used for a duplicate GeneXpert assay and 100 μL was used for DNA extraction and real-time MPX-generic PCR. At CDC, DNA was extracted from the supernatant using the EZ1 DNA Tissue Kit (Qiagen) in conjunction with the EZ1 platform (Qiagen).
All steps requiring specimen manipulation, including swab processing, crust homogenization, elution in PBS, and transfer to the GeneXpert cartridge were conducted within a certified biosafety cabinet.
4
Colorectal Cancer Tissue Collection and DNA Extraction
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We obtained matched primary CRC, pre-FOLFOX metastasis, post-FOLFOX metastasis, and normal colorectal tissue samples from four patients. The normal colorectal tissues were collected from surgical specimens of the primary tumors. All tissue samples were formalin-fixed, paraffin-embedded (FFPE) specimens. DNA samples were obtained from macroscopically dissected FFPE specimens cut into 10-μm-thick sections. Genomic DNA was extracted using EZ1 Advanced XL and EZ1 DNA Tissue Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions [16 (link)]. Nucleic acid yields were determined using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and the quality of genomic DNA was examined using a Quant-iT picoGreen dsDNA (Life Technologies, Carlsbad, CA, USA) assay kit.
5
Leptospira Isolation and Identification
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In addition to the sampling and storage at -80°C of organs and urine mentioned above, a few urine droplets and/or a small piece of freshly sampled kidney (crushed under sterile conditions) were used individually to inoculate three distinct culture media: (i) Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium (Difco, Detroit, MI, USA) supplemented with Albumin Fatty Acid Supplement (AFAS; Royal Tropical Institute, Amsterdam, Netherlands) [10 (link)] (ii) EMJH liquid medium supplemented with AFAS, rabbit serum and fetal calf serum (1% each); and (iii) semisolid Fletcher medium (Difco, Detroit, MI, USA) supplemented with rabbit serum (8%). All media were supplemented with 5-fluorouracil (5-FU) at a final concentration of 200 μg.mL-1. Cultures were incubated at 28°C, visually checked for the presence of leptospires using a dark field microscope once a week for four months and positive cultures were further sub-cultured in fresh EMJH liquid medium deprived of 5-FU. DNA was extracted from 1 mL of each positive culture using the EZ1 Biorobot with Qiagen EZ1 DNA Tissue kits (Qiagen, Les Ulis, France).
6
Leptospira Isolation from Kidney Cultures
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Twenty-four animal samples were randomly selected in order to attempt Leptospira isolation from kidney cultures, i.e. 15 R. rattus, two R. norvegicus, two M. musculus, three S. murinus and two T. ecaudatus. A small piece of the freshly sampled kidney was crushed under sterile conditions and used to inoculate three distinct culture media: (i) Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium (Difco, Detroit, MI, USA) supplemented with Albumin Fatty Acid Supplement (AFAS; Royal Tropical Institute, Amsterdam, Netherlands) [38 (link),39 (link)]; (ii) EMJH liquid medium supplemented with AFAS, rabbit serum and foetal calf serum (1% each); and (iii) semisolid Fletcher medium (Difco, Detroit, MI, USA) supplemented with rabbit serum (8%). All media were supplemented with 5-fluorouracil (5-FU) at a final concentration of 200 μg.mL-1. Cultures were incubated at 28°C, visually checked for the presence of Leptospira using a dark field microscope once a week for four months, and positive cultures were further sub-cultured in fresh EMJH liquid medium deprived of 5-FU. DNA was extracted from 1 mL of each positive culture using the EZ1 Biorobot with Qiagen EZ1 DNA Tissue kits (Qiagen, Les Ulis, France).
7
Phylogenetic Typing of ESBL-E Isolates
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All the ESBL-E isolates identified in the long-term carriers at each time point were studied. DNA was extracted using the EZ1 DNA tissue kit (Qiagen, Courtaboeuf, France). The phylogenetic group of E. coli Escherichia
8
Optimized Microbial DNA Extraction
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DNA was extracted from 0.1 ± 0.03 g wet faeces using the QIAamp PowerFecal DNA kit on a QIAcube (Qiagen, Hilden, Germany) as described previously [14 (link)].
DNA was extracted from cultured L. reuteri with the EZ1 DNA Tissue kit (Qiagen). Broth cultures were centrifuged at 3000 rpm for 10 min at 4 °C, and pellets were resuspended in 500 µL buffer G2, while up to 10 colonies were picked from agar plates and pooled in 500 µL G2 buffer. Samples were transferred to glass bead tubes and shaken with a TissueLyser II for 1 min at 30 Hz. Two hundred µL lysate were subjected to automatised DNA extraction using the protocol for purification of DNA from bacterial culture samples on an EZ1 Advanced XL robot (Qiagen).
DNA concentration was measured with Qubit dsDNA HS Assay kits (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, and DNA was stored at −20 °C.
DNA was extracted from cultured L. reuteri with the EZ1 DNA Tissue kit (Qiagen). Broth cultures were centrifuged at 3000 rpm for 10 min at 4 °C, and pellets were resuspended in 500 µL buffer G2, while up to 10 colonies were picked from agar plates and pooled in 500 µL G2 buffer. Samples were transferred to glass bead tubes and shaken with a TissueLyser II for 1 min at 30 Hz. Two hundred µL lysate were subjected to automatised DNA extraction using the protocol for purification of DNA from bacterial culture samples on an EZ1 Advanced XL robot (Qiagen).
DNA concentration was measured with Qubit dsDNA HS Assay kits (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, and DNA was stored at −20 °C.
9
DNA Extraction from Blood and Ticks
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DNA extraction was performed on a Bio Robot EZ1 (Qiagen, Courtaboeuf, France) using a commercial EZ1 DNA Tissue Kit (Qiagen) according to the manufacturer’s instructions. DNA was extracted from 200 µl of blood from all the animal samples. Ticks were recovered from ethanol, rinsed with distilled water and dried on sterile filter paper in a laminar-flow hood. Each tick was cut in half lengthways (the blades were discarded after each tick was cut). DNA was individually extracted from one-half, and the remaining tick halves were frozen at − 80 °C for subsequent studies as previously described [22 (link)].
10
Mosquito Genomic DNA Extraction and Wolbachia Detection
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Genomic DNA was extracted from the abdomen of individual mosquito using the Biorobot EZ1 System with the EZ1 DNA tissue kit [Qiagen, Court a boeuf, France] following the manufacturer’s instructions. Individual mosquitoes were screened as described in Shaw et al. [18 (link)] using both the standard [W16S-Spec] [24 (link)] and the nested PCR [W16S-Nested] [17 (link)] protocols. A third qPCR assay [W16S-qPCR] recently developed [10 (link)] was used to confirm Wolbachia infection in An. funestus.
The nested-16S rDNA Wolbachia primers were used to generate a 340–412-bp fragment according to Baldini et al. [17 (link)]. The PCR products of all Wolbachia-positive samples were purified by filtration using NucleoFast® 96 PCR DNA purification plate then amplified using the BigDye™ Terminator v3.1 Cycle Sequencing Kit [Applied Biosystems, Foster City, CA]. The BigDye PCR products were purified on the Sephadex G-50 Superfine gel filtration resin prior the sequencing on the ABI Prism 3130XL.
The nested-16S rDNA Wolbachia primers were used to generate a 340–412-bp fragment according to Baldini et al. [17 (link)]. The PCR products of all Wolbachia-positive samples were purified by filtration using NucleoFast® 96 PCR DNA purification plate then amplified using the BigDye™ Terminator v3.1 Cycle Sequencing Kit [Applied Biosystems, Foster City, CA]. The BigDye PCR products were purified on the Sephadex G-50 Superfine gel filtration resin prior the sequencing on the ABI Prism 3130XL.
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