Tissue samples were thawed at room temperature, and then 25–30 mg of tissue was cut and placed in Precellys CK28 tubes containing Qiazol Lysis buffer (Qiagen, Hilden, Germany). These tissue samples were disrupted and homogenized at room temperature using the 5000:3×20 - 20 s wait program on the Precellys 24 (Bertin, Rockville, MD, USA). Next, the supernatant was transferred into new 2-mL tubes (Eppendorf, Hamburg, Germany) and 200 µL chloroform was added. This mixture was incubated at room temperature for 2 min, and then the tubes were centrifuged at 11.600× g at 4 °C for 15 min. The upper aqueous phase was collected and transferred to 2-mL tubes (Eppendorf), and total RNA was isolated using the miRNeasy Mini kit (Qiagen, Hilden, Germany), including a DNase I digestion, following the manufacturer’s protocol. Total RNA quality and quantity were assessed using the Nanodrop One (Thermo Fisher Scientific).
2 ml tube
Manufactured by Eppendorf
Sourced in Germany
The 2 mL tubes are versatile laboratory containers designed to hold small volumes of liquids or powders. They provide a compact and reliable storage solution for a variety of sample types in research and diagnostic settings.
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Lab products found in correlation
Z 8200, by Hitachi (3 mentions)
Tissuelyser 2, by Qiagen (3 mentions)
Suprapure, by Merck Group (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Sv total rna isolation system, by Promega (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Qiazol lysis buffer, by Qiagen (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
Ammonia water, by Fujifilm (1 mentions)
Micromixer e 36, by TAITEC (1 mentions)
Br 23fp, by TAITEC (1 mentions)
Vacutainer, by BD (1 mentions)
Sodium citrate, by Thermo Fisher Scientific (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
5 mm steel bead, by Qiagen (1 mentions)
Heparin tube, by BD (1 mentions)
Phosphate buffered saline (pbs), by Biosesang (1 mentions)
Xradia 520 versa x ray microscope, by Zeiss (1 mentions)
Celltrics, by Sysmex (1 mentions)
33 protocols using 2 ml tube
1
Tissue RNA Extraction Protocol
2
Quantitative Hair Steroid Analysis
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The impact of contamination by sweat, sebum, or excreta may be minimal if samples are washed [15 (link),19 (link),20 (link)]. The hair washing and steroid extraction procedures were based on the protocol described in Stalder et al. [26 (link)] (study II) with modifications made by Gao et al. [27 (link)] to allow analysis by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS): Hair strands were washed by shaking them in 2.5 mL isopropanol for 3 min at room temperature. They were then allowed to dry under a fume hood for at least 12 h. A total of 10 mg of whole, non-pulverized hair was carefully weighed and transferred into a 2 mL tube (Eppendorf AG, Hamburg, Germany). After this, 50 μL internal standard and 1.8 mL methanol were added, and the hair was incubated for 18 h at room temperature for steroid extraction. Samples were spun in a centrifuge at 10,000 rpm for 2 min and 1 mL of the clear supernatant was transferred into a new 2 mL tube. The alcohol was evaporated at 65 °C under a constant stream of nitrogen until the samples were completely dried (duration: approximately 20 min). The dry residue was re-suspended using 250 μL distilled water, 200 μL of which was used for LC–MS/MS analysis [27 (link)].
3
Extracting Epiphytic Communities from Plants
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To obtain epiphytic communities all plant samples were run through a washing procedure by shaking (vortex) for 3 min in sterile 1x PBS-Silwet buffer; pH 7.4 (Silwet L77, final conc. 0.02%), followed by ultrasonication (640 W, Sonorex Digipuls, DL 510 H, Bandelin, Berlin, Germany) for 3 min. The buffer volumes were 100 ml for Achillea and 200 ml for Hamamelis, respectively. The sonication power was reduced to 80% (512 W) for young leaves from April to minimize cell damage. The washed-off epiphytic fraction from 2016 (time series) was centrifuged first in a 50 ml Falkon tube for 15 min at 4,369 × g followed by 12,900 × g in 2 ml tube (Eppendorf, Hamburg, Germany). In 2018 washed-off epiphytic fractions (spatial comparison) were filtered through a sterile 0.2 μm filter and stored at −80°C until DNA extraction. The endophytic plant samples, after ultrasonication treatment, were transferred in 50 ml Falkon tubes, rinsed additionally by shaking in fresh sterile PBS buffer to remove remaining epiphytes and stored at −80°C until extraction. For each extraction, PBS-Silwet buffer served as control.
4
Recombinant Protein and Amyloid Fiber Formation
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Hamster recombinant PrP used for TEM observation and mouse recombinant PrP used for CD spectrum measurement were dissolved in PBS and 5 mM Tris–HCl (pH 7.5), respectively, and allowed to form fibers by standing. Aβ-peptide (Human,1–40) (HCl Form) (Peptide Institute, Osaka, Japan) was dissolved in 0.05% ammonia water (Fujifilm Wako, Osaka, Japan) to 500 µM. Aβ (1–40) fibrils used for TEM observation were diluted by adding 50 mM sodium phosphate (pH 7.5) to a concentration of 100 µM. 150 µl of the solution was added to a 2 ml tube (Eppendorf AG, Hamburg, Germany) and shaken at 1,500 rpm for 16 h at room temperature using a MicroMixer E-36 (TAITEC CORPORATION, Saitama, Japan) to form fibers. For the samples used for CD spectra, Aβ (1–40) was diluted to 100 µM using PBS, and then 300 µl of the solution was added to a centrifuge tube. The sample was shaken at 250 rpm for 12 h at 37 °C using a BR-23FP (TAITEC CORPORATION, Saitama, Japan).
5
Plasma Fractionation for Biomarker Analysis
6
Hair Cortisone Extraction and Analysis
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Hair samples were prepared for the cortisone assay following standard methods described by Gao et al. (2013) . Specifically, a minimum of 7·5 mg of hair was obtained from each hair sample ≥ 3 cm. Hair samples shorter than 3 cm were not assayed. The hair washing and cortisone extraction procedures were based on the protocol previously described in Stalder et al. (2012) (link). In brief, hair strands were washed by shaking them in 2·5 mL isopropanol for 3 min at room temperature and then dried under a fume hood for at least 12 h. 7·5 mg of whole non-pulverised hair was then carefully weighed out and transferred into a 2 mL tube (Eppendorf, Hamburg, Germany). 50 μL internal standard and 1·8 mL methanol were added and the hair was incubated for 18 h at room temperature for cortisone extraction. Samples were spun in a centrifuge at 10,000 rpm for 2 min and the clear supernatant was transferred into a new 2 mL tube. The alcohol was evaporated at 65 ◦C under a constant stream of nitrogen for approximately 20 min until the samples were completely dried. The dry residue was resuspended using 250 μL distilled water, 200 μL of which was used for liquid chromatography tandem mass spectrometry (LC-MC) analysis. More details of the protocol are provided by Gao et al. (2013) .
7
Optimized Extraction of Bioactive Compounds
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Thirty milligrams of each lyophilized sample and 5 mm steel bead (QIAGEN) were placed in a 2 mL tube (Eppendorf) and ground to homogeneous fine powder by Tissue-Lyser (QIAGEN) in the frequency of 30 Hz for 3 min. For the comparative analysis in different blooming stages, those milled samples were added to 1 mL of the solvent established through mixture design and extracted according to the optimal shaking extraction conditions from the 23 full factorial design and polynomial regression analysis.
8
Isolation and RNA Extraction of PBMCs
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For the analysis, PBMCs were stored at room temperature in heparin tubes (Becton-Dickinson, Belliver Industril Estate, UK). We transferred 3 mL of Histopaque-1077 (RNBF8871, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) and 3 mL of blood to a 15 mL conical tube. Subsequently, we centrifuged the mixture at 400× g for 30 min at 20 °C and extracted 2 mL of the white buffy coat layer. Only the buffy coat was placed in a 15 mL tube containing 8 mL of PBS (P2004, Biosesang, Yongin, South Korea), which was centrifuged at 300× g for 10 min at 20 °C. After the centrifugation, we removed the supernatant, leaving the pellet that had settled to the bottom, to which we added 1 mL of Trizol (TR 118, Molecular Research Center, Cincinnati, OH, USA) to release the pellet, which we transferred to a 2 mL tube (Eppendorf AG, Hamburg, Germany).
9
Quantifying Quadriceps Muscle Mineralization
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Animals were harvested at 8 weeks of age. The left quadriceps muscle was fixed in 10% formalin for 48 h, then to transferred to 70% ethanol prior to scanning. Quadriceps were placed within a 2-ml tube (Eppendorf, Stevenage, United Kingdom) and supported by a polyurethane foam saturated in 70% ethanol. Muscles were imaged using a Zeiss Xradia 520 Versa X-ray microscope (Zeiss) operating at an energy of 50 kV, a power of 4 W, and a tube current of 80 μA with a Zeiss LE1 filter was positioned directly after the X-ray source. The sample distance to the x-ray source was 200mm, and the sample distance to detector was 225mm, A total of 1601 X-ray projection images were collected over 360° at equal intervals with an exposure time for each projection of 3 s. The projections were reconstructed using the manufacturer’s integrated software, which uses a filtered back projection reconstruction algorithm. TXRM files were imported into FiJi with the plugin to read XRM files and export Z-stack images (Schindelin et al., 2012 (link); Metscher 2020 ). Mineralised areas within exported z-stack images (pixel size ranging from 32.25 to 34.45 µM depending upon sample) were quantified using the threshold function in ImageJ and used to calculate the volume.
10
Genomic DNA Extraction from Tissue Samples
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A 50-100 mg sample of tissue cut up into pieces was added to a preheated (56 °C) 2 mL tube (Eppendorf) loaded with 1 mL lysis buffer (100 μL 20 mg/mL proteinase K and 100 μL 20% sodium dodecyl sulfate) and incubated at 56 °C for 60-120 minutes. After cooling to room temperature, the tube was centrifuged at 18213 × g for 10 minutes to obtain the supernatant. The supernatant was transferred to a new 2.0 mL tube, an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) added, and the tube centrifuged at 18213 × g for 10 minutes. The supernatant was transferred to a new 1.5 mL tube and isopropyl alcohol to 2/3 volume of the supernatant added (1/10 volume of 3M sodium acetate was added if necessary), the tube was inverted at least 3 times and placed at -20 °C for 2 hours for precipitation. To remove the supernatant, the tube was centrifuged at 18213 × g for 10 minutes to obtain the DNA pellet which was then washed with 1 mL 75% ethanol. The pellet was resuspended by centrifuging at 18213 × g for 5 minutes at room temperature and the supernatant removed. The DNA pellet was air-dried in a biosafety cabinet for a few minutes and 25-100 μL of TE buffer added to dissolve the DNA pellet.
The DNA concentration was detected by Qubit Fluorometer. Sample integrity and purity were detected by agarose gel electrophoresis.
The DNA concentration was detected by Qubit Fluorometer. Sample integrity and purity were detected by agarose gel electrophoresis.
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