An input mass of 1 μg of DNA was used for each sample for library preparation, following the instructions of ONT 1D Native barcoding genomic DNA protocol from May 2018. The following procedures complemented the ONT protocol: DNA repair was carried out using the NEBNext Companion Module (New England Biolabs). This step was followed by the barcode ligation using NEB Blunt/TA Ligase (New England Biolabs) and EXP-NBD104 native barcode kit (ONT). After barcoding, 10-12 DNA samples were pooled resulting in a total of 1 μg DNA. For adapter ligation, the NEBNext Quick Ligation Module (New England Biolabs) and SQK-LSK109 ligation sequencing kit (ONT) were used. Throughout the protocol, AMPure XP beads (Beckman Coulter) were used to purify the samples. After DNA repair and barcode ligation the Elution Buffer (Promega Corporation) was selected for DNA Elution. Following adapter ligation, the DNA was eluted with Elution Buffer of SQK-LSK109 ligation sequencing kit (ONT). MinION sequencing was performed on the SpotON Flow Cell with the Flow Cell Priming Kit (Flow Cell Type R9.4.1, ONT). On average, ~250 ng of library DNA were loaded onto the flow cell for one run.
Elution buffer
Manufactured by Promega
Elution Buffer is a solution used to elute or release target molecules from a solid support or column during a purification process. It provides the necessary conditions to efficiently recover the desired product from the purification matrix.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Nebnext companion module, by New England Biolabs (1 mentions)
Neb blunt ta ligase, by New England Biolabs (1 mentions)
Nebnext quick ligation module, by New England Biolabs (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Nanodrop fluorometer, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Promega (1 mentions)
Maxwell 16 lev blood dna kit, by Promega (1 mentions)
Quantus fluorometer, by Promega (1 mentions)
Blood dna kit, by Promega (1 mentions)
Lysis buffer, by Promega (1 mentions)
Maxwell rsc purefood gmo and authentication kit, by Promega (1 mentions)
Ctab buffer, by Promega (1 mentions)
Maxwell rsc instrument, by Promega (1 mentions)
6 protocols using elution buffer
1
ONT Native DNA Barcoding Protocol
2
DNA Extraction from Frozen Tissue Sections
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DNA was isolated from 20 to 40 frozen tissue sections per sample (10 μm) from 39 cases, taken adjacent to sections used for tumor content assessment, as per 100 000 Genomes Project standard operating procedures. The Maxwell 16 LEV Blood DNA Kit (AS1290; Promega) was used to extract DNA. Before extraction samples were homogenized by the addition of 300 μL lysis buffer and 30 μL Proteinase K (Promega) to each sample, followed by incubation at 56°C for at 30 minutes. Samples were then transferred to Maxwell 16 LEV Cartridges for extraction, according to the manufacturer's protocol. DNA was eluted in 70 μL Elution Buffer (Promega). Concentration was assessed using the Qubit dsDNA HS Assay Kit with the Qubit Fluorometer (Thermo Fisher Scientific) and 260:280 ratio assessed using the Nanodrop Fluorometer (Thermo Fisher Scientific) according to manufacturer's instructions.
3
Comprehensive Hylidae Phylogenetic Analysis
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To achieve phylogenetic representation across Hylidae, we selected three species (26 total samples) from each of the subfamilies: Acrisinae, Hylinae (Holarctic and Middle American), Pseudinae, Dendropsophinae, Lophyohylinae, Scinaxinae, and Cophomantinae. We also selected one species each from the families Phyllomedusidae and Pelodryadidae to be used as outgroups. The UCE data from 7 samples are first published in Portik et al., in press as part of a large UCE phylogeny of all frogs. Tissue samples for molecular work were obtained from the museum holdings of the University of Kansas (KU), California Academy of Science (CAS), Museum of Vertebrate Zoology at Berkeley (MVZ), and Museo de Zoología Universidad Technologica Indoamérica, Quito, Ecuador (MZUTI). Sample metadata are included as supplementary table S1, Supplementary Material online. Genomic DNA was extracted from the tissue samples with a PROMEGA Maxwell bead extraction robot. The resultant DNA was quantified using a PROMEGA Quantus Fluorometer. Approximately 500 ng total DNA was acquired and set to a volume of 50 μl through dilution with Promega elution buffer or concentration using a vacuum centrifuge when over 50 μl.
4
Genomic DNA Extraction from Animal Tissue
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We obtained genomic DNA extracted from muscle or liver with the ReliaPrep™ (Promega Madison, WI, USA) gDNA Tissue kit, using the standard protocol for animal tissue. We extracted and purified blood samples using phenol-chloroform. These samples were prepared by breaking 1–2 cm of the glass capillary containing the blood (∼4 μl) and placing it overnight at 56°C in a 2 ml tube containing: lysis buffer B (400 mM Tris-HCl, pH 8.0; 100 mM EthyleneDiamine Tetra-Acetic acid [EDTA], pH 8.0; 1% Sodium Dodecyl Sulfate [SDS]), 250 µl of TBS buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl), and 40 µl of Proteinase K (20 mg/ml). Samples from Spain and Nebraska were instead extracted with magnetic beads on a 16 Maxwell® RSC 16 instrument using the dedicated Blood DNA Kit (Promega) and employing the “Blood DNA” protocol. Sample preparation in this case was performed by adding 1–2 μl of blood to 300 μl of lysis buffer and 30 μl Proteinase K and incubating overnight at 56°C. Genomic DNAs were eluted into TE buffer (10 mM Tris-Cl, 1 mM EDTA) or elution buffer (Promega).
5
Bacterial DNA Extraction from River Water
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For each extraction, 90 ml of river water were filtered through a 0.2 µm filter using a vacuum pump. Then, the bacteria on the filter were resuspended in 1 ml of CTAB Buffer (Promega, Madison, Wisconsin, USA) and heated for 5 min at 95 °C. After vortexing for 1 min, 300 µl of supernatant were added to 300 µl of Lysis Buffer (Promega) and purified using the Maxwell® RSC Instrument and the Maxwell® RSC PureFood GMO and Authentication Kit (Promega). DNA was eluted in 100 µl of Elution Buffer (Promega).
6
Plasma cfDNA Extraction Protocol
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cfDNA was extracted using the Maxwell RSC LV ccfDNA kit (Promega). Isolation of cfDNA was done starting from 1500 µL of plasma. DNA extraction was performed according to the manufacturer’s instructions. DNA was eluted in 75 μL of elution buffer (Promega).
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