One µg of Prostaglandin E2-d4 (CAYMAN CHEMICAL, Michigan, USA) was added as internal standard to the media (100–200 mL) recovered from the cell culture, from day 3 to day 10, through a filtration step on 1.2 µm nitrocellulose filters (Millipore RAWP04700). Culture media were loaded onto pre-packed CHROMABOND® HR-X cartridges (500 mg/6 mL) previously activated with methanol (12 mL) and milliQ water (12 mL). After a preliminary desalting step with 12 mL of milliQ water, collection of the organic components was achieved by elution with 16 mL of methanol followed by 16 mL of methanol/dichloromethane (1:1). The two organic fractions were combined and dried under reduced pressure for LC-MS/MS analysis.
Rawp04700
Manufactured by Merck Group
Sourced in United States
RAWP04700 is a laboratory equipment product from Merck Group. It is designed for use in scientific research and analysis. The core function of this product is to provide accurate and reliable measurement and data collection capabilities for laboratory applications. No further details about the intended use or specific features of this product can be provided in an unbiased and factual manner.
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Lab products found in correlation
4 protocols using rawp04700
1
Prostaglandin E2-d4 Extraction and Analysis
2
Genome Assembly of Pseudo-nitzschia multistriata
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B856 cells were collected onto 1.2‐μm pore‐size membrane filters (RAWP04700 Millipore) and DNA was extracted with phenol–chloroform as described in Sabatino et al. (2015 ). The P. multistriata genome was assembled from a total of 172 million 101‐bp overlapping paired‐end reads with c. 175‐bp inserts, 117 million 100‐bp paired‐end reads with c. 450‐bp inserts, 72 million c. 68‐bp (after trimming) mate pair reads with c. 1.2‐kb inserts and 5.4 million c. 156‐bp (after trimming) mate pair reads with c. 4.5‐kb inserts. Mate pair libraries were processed by Next Clip to remove adapters. Depending on the library, the genome size was estimated to be between 71 and 82 Mb using SGA preqc. Reads from libraries exceeding 100× coverage were randomly subsampled to 100× and then assembled into scaffolds by Allpaths ‐LG (Gnerre et al., 2011 ) via Rampart (Mapleson et al., 2015 ). The completeness of the genome was evaluated using Cegma with the set of 248 core eukaryotic genes (CEGs) (Parra et al., 2007 ). The assembly (accession number PRJEB9419) can be visualized at http://apollo.tgac.ac.uk/Pseudo-nitzschia_multistriata_V1_4_browser/sequences (username and password: pnitzschia).
3
Spore Disaggregation and Virus Quantification
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Cultures with microscopically‐verified spores were sonicated three times on ice for 1 min with a Branson 250 sonicator (100 W; Branson, Emerson Electric Co., USA) to break up vegetative cells into smaller pieces, and then filtered onto 1.2 μm pore‐size polycarbonate filters (RAWP04700; Millipore). Filters were washed three times with 50 ml of sterile ASW to remove any free viruses and cell debris. Samples were collected before and after washing and analysed via the most probable number (MPN) assay to determine the number of infectious viruses. A subsample was also collected to measure the concentration of clean spores.
4
Isolation and Culture of Thalassiosira rotula
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Thalassiosira rotula, strain FE80C, was isolated in 2011 in the Gulf of Naples (40°48.5’N, 14°15’E), Mediterranean Sea. Clonal cultures were established by isolating single cells or short chains from phytoplankton net samples collected from the surface layer of the water column. Cultures were grown in sterile filtered oligotrophic seawater at 36 psu amended with f/251 nutrients and maintained at a temperature of 20 °C, at 12:12 h light:dark cycle, and with a photon flux of 100 μmol photons m-2s−1.
10-liter cultures of T. rotula, in triplicate, were used to follow their growth from day 3 to day 10. Every day, 250 mL of each culture was harvested by filtration onto 1.2 μm pore size filters (RAWP04700 Millipore) and immediately frozen in liquid nitrogen. 100–200 mL of culture media recovered from the cell filtration was collected and stored at −80 °C until sample processing. Initial cell concentrations were about 5000 cells/mL upon inoculation. Culture growth was monitored daily from samples fixed with one drop of Lugol (final concentration of about 2%) and counted in a Bürker counting chamber under an Axioskop 2 microscope (20×) (Carl Zeiss GmbH, Jena, Germany).
10-liter cultures of T. rotula, in triplicate, were used to follow their growth from day 3 to day 10. Every day, 250 mL of each culture was harvested by filtration onto 1.2 μm pore size filters (RAWP04700 Millipore) and immediately frozen in liquid nitrogen. 100–200 mL of culture media recovered from the cell filtration was collected and stored at −80 °C until sample processing. Initial cell concentrations were about 5000 cells/mL upon inoculation. Culture growth was monitored daily from samples fixed with one drop of Lugol (final concentration of about 2%) and counted in a Bürker counting chamber under an Axioskop 2 microscope (20×) (Carl Zeiss GmbH, Jena, Germany).
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