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Authors: Scott Edmunds, Jesse Xiao, Hongling Zhou
Group: BGI
Summary: This protocol is for teachers to set up a data curation exercise for their students. Using a universities IR or CRIS to select papers that their researchers have published, and then assign them to students to see if the data availability meets the national, funder, university and journal policies. The students carrying out this assessment have a separate related protocol they need to follow.

Proper citation: Scott Edmunds, Jesse Xiao, Hongling Zhou 2019. University Published Paper Reproducibility Assessment Protocol - For Teachers. protocols.io https://dx.doi.org/10.17504/protocols.io.6x8hfrw Copy   

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Authors: Sade Aisha Folashade John, Patrick E. Akpaka, Chandrashekhar Unakal, Arvind Kurhade, Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Enterobacteriaceae the leading cause of gastroenteritis. These gram- negative bacteria from species like Escherichia, Shigella, Salmonella, Vibrio and Helicobacter etc are among those that cause severe disease in consumers, especially those that indulge in uncooked seafood. Oysters have been shown to harbour these pathogenic organisms, which pose a major health risk. Numerous outbreaks of gastroenteritis due to oyster consumption have been reported worldwide because of the enteric bacteria as well as viral pathogen contaminationThis research was conducted to identify the Enterobacteriaceae present in oysters sold by vendors in Trinidad. During a five-month period (the rainy season, May - September), a total of 156 oyster samples (comprising 104 oysters samples and 52 water samples of the prepared, unprepared oyster cocktails plus the water used by vendors) were analysed. These samples were collected from roadside vendors operating near to the coastal line of the Western part of Trinidad These were processed at the microbiology unit of the Department of Para-clinical Sciences of the University of the West Indies, St. Augustine using standard microbiology techniques. The antibiotic susceptibility profiles of the recovered organisms were performed using the Kirby Bauer method for the following antibiotics- Tetracycline, Ampicillin, Trimethoprim-Sulfamethoxazole, Cefuroxime and Ceftazidime. The SPSS 21 programme was used to analyse the biometric data and a chi-square test was used to determine if there was any significant difference between the kinds of preparation cocktails with respect to the coliform and organisms found.Results of the 104 oyster samples tested, 112 isolates were obtained, 13.4% (15/112) were E.coli of which 0.1% (1/15) was the 0157:H7 strain; 72.3% (81/112) were Shigella species of which 27.2% (22/81) were Shigella dysenteriae; 14.3% (16/112) were Salmonella species of which 25% (4/16) were Salmonella cubana. The susceptibility profiles of the organisms revealed that 86.5% of Shigella, 100% of E.coli and 93.7% of the Salmonella were resistant to multiple antibiotics. With the recovery of these organisms from these samples, the health of the Trinidadian and foreign consumers of mangrove oysters is at risk particularly from Shigella, E.coli and Salmonella species. As a result, there should be an increased need for public awareness as well as regulations by the Ministry of Health to be made so that illness from these hazardous Enterobacteriaceae can be prevented.

Proper citation: Sade Aisha Folashade John, Patrick E. Akpaka, Chandrashekhar Unakal, Arvind Kurhade, Angel Justiz-Vaillant 2020. Detection of total and faecal coliforms in the oysters. protocols.io https://dx.doi.org/10.17504/protocols.io.bj6hkrb6 Copy   

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Authors: Rodney Pettway
Group: Zymoseptoria community protocols (STBnet)
Summary: Preparation of Zymoseptoria tritici long-term storage on silica gel. Rodney Pettway 1992, ed. Petteri Karisto 2018.

Proper citation: Rodney Pettway 2018. Long-term storage of Zymoseptoria tritici on silica gel. protocols.io https://dx.doi.org/10.17504/protocols.io.s2regd6 Copy   

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Authors: Anton Du Plessis
Summary: The use of microCT of 10 mm coupon samples produced by AM has the potential to provide useful information of mean density and detailed porosity information of the interior of the samples. In addition, the same scan data can be used to provide surface roughness analysis of the as-built surfaces of the same coupon samples. This can be used to compare process parameters or new materials. While surface roughness is traditionally done using tactile probes or with non-contact interferometric techniques, the complex surfaces in AM are sometimes difficult to access and may be very rough, with undercuts and may be difficult to accurately measure using traditional techniques which are meant for smoother surfaces. This standard workflow demonstrates on a coupon sample how to acquire surface roughness results, and compares the results from roughly the same area of the same sample with tactile probe results. The same principle can be applied to more complex parts, keeping in mind the resolution limit vs sample size of microCT.

Proper citation: Anton Du Plessis 2018. Standard method for microCT-based additive manufacturing quality control 3: surface roughness. protocols.io https://dx.doi.org/10.17504/protocols.io.sbweape Copy   

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Authors: Xiling Shen, Marcos Negrete, Kun Xiang
Group: NCI PDMC consortium
Summary: This protocol is split into 3 sections: collecting tumor cells, passaging, and cryopreserving organoids. Phase 1: Aim:Collect viable cells from biopsy samples Tumor organoid: Colorectal CancerSource: Human primary tissue Phase 2:Aim: Passage and expand organoid samplesPhase 3:Aim: Organoid cryopreservationTumor organoid: Colorectal CancerSource: Human primary tissue

Proper citation: Xiling Shen, Marcos Negrete, Kun Xiang 2020. Duke - Isolation, Culture, and Maintenance of Patient-Derived Tumor Biopsy. protocols.io https://dx.doi.org/10.17504/protocols.io.bijikcke Copy   

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Authors: Guanshi Zhang, Dusan Velickovic, Annapurna Pamreddy, Jessica Lukowski, Theodore Alexandrov, Chris Anderton, Kumar Sharma
Group: KPMP, Metabolomics Protocols & Workflows
Summary: Mass spectrometry imaging (MSI) is a cutting-edge molecular technology that enables simultaneous analysis of multiple molecular components directly from single cells, tissues, and organs. For MSI, cryosections are prepared from flash-frozen tissue and mounted on a contuctive glass slide. We use matrix-assisted laser desorption ionization (MALDI)-MSI, where tissue sections are coated with a MALDI matrix in order to facilitate laser ionization and detection of metabolites with mass spectrometry. We do it using an automated robotic sprayer (TM-Sprayer) with 2,5- dihydroxybenzoic acid (DHB) MALDI matrix for positive ion mode analysis, N-(1-naphthyl) ethylenediamine hydrochloride (NEDC) for negative ion mode analyses, or 1,5-Diaminonaphthalene (DAN) for dual-polarity analysis, prior to being loaded into the MALDI-Q Exactive HF-X Orbitrap-MSI or MALDI-FTICR-MSI for untargeted metabolomics analysis. We have optimized a method for matrix application to maximize analyte extraction from tissue and increase MALDI sensitivity and to create the most homogenous matrix films possible for best lateral resolution of lipids. A key capacity that will be critical for scale-up in KPMP is the use of a TM-sprayer robotic sprayer for MALDI matrix application. This will make inter-lab studies viable, by increasing the reproducibility of sample preparation and is currently in use at UTHSA and PNNL. Our lipid extraction protocol for LC-MS/MS is a robust and universal protocol for single-sample integrative proteomics, metabolomics, and lipidomics analyses (see citation).

Proper citation: Guanshi Zhang, Dusan Velickovic, Annapurna Pamreddy, Jessica Lukowski, Theodore Alexandrov, Chris Anderton, Kumar Sharma 2020. Tissue Preparation for Spatial Metabolomics. protocols.io https://dx.doi.org/10.17504/protocols.io.bcraiv2e Copy   

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Authors:
Group: UCSC BME 22L

Proper citation: 2020. How to Label a Gel. protocols.io https:// Copy   

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Authors: SANTIAGO SEPULVEDA

Proper citation: SANTIAGO SEPULVEDA 2020. QIAamp DNA FFPE Tissue Kit protocol. protocols.io https:// Copy   

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Authors: Gregory M. Mueller, John Paul Schmit, Sabine M. Huhndorf, Leif Ryvarden, Thomas E. O'Dell, D. Jean Lodge, Patrick R. Leacock, Milagro Mata, Loengrin Umana, Qiuxin (Florence) Wu, and Daniel L. Czederpilz, Daniel L. Czederpiltz
Summary: The protocol for sampling conspicuous macrofungi using fixed-size plots.

Proper citation: Gregory M. Mueller, John Paul Schmit, Sabine M. Huhndorf, Leif Ryvarden, Thomas E. O'Dell, D. Jean Lodge, Patrick R. Leacock, Milagro Mata, Loengrin Umana, Qiuxin (Florence) Wu, and Daniel L. Czederpilz, Daniel L. Czederpiltz 2019. Sampling macrofungi using fixed-sized plots. protocols.io https://dx.doi.org/10.17504/protocols.io.z2xf8fn Copy   

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Authors: Jakub Nedbal
Summary: Microalgae are grown for the research on photosynthesis, biotechnology, and water-environment ecology. Specialized laboratories typically use calibrated commercial equipment, which agitates the microalgae culture by shaking or bubbling, controls the irradiance, temperature, and CO2 content. Such commercial incubators may be out of reach for some laboratories, like those not specializing in microalgae research, teaching laboratories, or laboratories in low resource settings. Our research uses microalgae cultures during the development and validation of advanced microscopy techniques. We lacked access to and the budget for a commercial incubator. We solved the problem by building a stand-alone orbital shaking incubator with an in-built variable light source and a 24-hour timer. The shaker features a homogeneously illuminated growth area of 20 cm × 15 cm, which is suitable for three T75 tissue culture flasks or four 100 ml Erlenmeyer flasks. The overall material cost was around £300 and assembly time of about two days. We tested the shaker with fresh-water microalgae Desmodesmus quadricauda and Chlorella vulgaris. Both microalgae cultures have been grown continuously for seven months in the incubator. We studied their growth under different light conditions to validate the function of the shaker. The protocol outlines the step-by-step process to the building of this microalgae shaker.

Proper citation: Jakub Nedbal 2020. Illuminated Orbital Shaker for Microalgae Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.8wuhxew Copy   

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Authors: Maria Rubio-Brotons
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab

Proper citation: Maria Rubio-Brotons 2016. Freezing and unfreezing unicellular protists using Mr Frosty container. protocols.io https://dx.doi.org/10.17504/protocols.io.gg3btyn Copy   

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Authors: Dr Ziyi Yu
Group: OpenPlant Project
Summary: A protocol used to generate a PDMS microfluidic chip. Work was funded by Cambridge Synthetic Biology Strategic Research Initative (SRI) SynBio Fund.http://www.synbio.cam.ac.uk/synbiofund

Proper citation: Dr Ziyi Yu 2016. PDMS Microfluidic Device Fabrication. protocols.io https://dx.doi.org/10.17504/protocols.io.ftybnpw Copy   

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Authors: Aliya Spigelman

Proper citation: Aliya Spigelman 2019. Static Glucose-stimulated Insulin Secretion (GSIS) Protocol - Human Islets. protocols.io https://dx.doi.org/10.17504/protocols.io.wy4ffyw Copy   

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Authors: Shivanthan Shanthikumar, Richard Saffery, Sarath C. Ranganathan, Melanie Neeland
Group: Respiratory Systems Immunology
Summary: This protocol describes the collection, processing, cryopreservation and thawing of pediatric bronchoalveolar lavage (BAL) samples for downstream single cell analysis (including flow cytometry, cell sorting, and single cell transcriptomics).

Proper citation: Shivanthan Shanthikumar, Richard Saffery, Sarath C. Ranganathan, Melanie Neeland 2021. Processing of pediatric bronchoalveolar lavage samples for single cell analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.btiankae Copy   

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Authors: Roey Angel, Eva Petrova, Ana Lara
Group: SoWa RI Anaerobic and Molecular Microbiology (public)
Summary: The following protocol describes how to perform an RNA-Stable Isotope Probing experiment. The scope of this protocol only covers the parts involving separating labelled RNA from unlabelled RNA using ultracentrifugation in a caesium trifluoroacetate density gradient and downstream quantification to evaluate whether the labelling and separation of the RNA were successful. Total RNA should be extracted from an environmental sample or an enrichment culture that was incubated with an isotopically-labelled substrate. Labelling can be of the carbon, oxygen or nitrogen in the RNA (or any combination of the 3). For environmental samples, we recommend extracting RNA using our protocol Total Nucleic Acids Extraction from Soil and purifying it using the Purification of RNA from Crude NA Extract protocol. This protocol is based on the following papers: Whiteley et al. (2007); Dumont et al. (2011); Angel and Conrad (2013). For a comprehensive discussion on how to design a SIP experiment and how to analyse the resulting data, we recommend referring to the recent book on the subject: Stable Isotope Probing: Methods and Protocols, especially chapters: 1-3 and 9-18.

Proper citation: Roey Angel, Eva Petrova, Ana Lara 2020. RNA-Stable Isotope Probing. protocols.io https://dx.doi.org/10.17504/protocols.io.bjaakiae Copy   

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Authors: Krystal Xia, Leo Jiang

Proper citation: Krystal Xia, Leo Jiang 2020. Determination of arsenic ion concentration. protocols.io https:// Copy   

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Authors: Brandon M. Satinsky, Scott M. Gifford, Byron C. Crump, Christina Smith, Mary Ann Moran
Group: Moran Lab
Summary: This protocol is from:Satinsky, Brandon M., et al. "Use of internal standards for quantitative metatranscriptome and metagenome analysis." Methods in enzymology 531 (2012): 237-250.Please see the full chapter for additional details.

Proper citation: Brandon M. Satinsky, Scott M. Gifford, Byron C. Crump, Christina Smith, Mary Ann Moran 2015. Preparation of Custom Synthesized RNA Transcript Standard. protocols.io https://dx.doi.org/10.17504/protocols.io.dxu7nv Copy   

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Authors: luanaborba
Group: L. P. Borba-Santos
Summary: Protocol that described the cytotoxicity assay using a mammalian epithelial cell line (LLC-MK2; ATCC CCL-7).

Proper citation: luanaborba 2020. Cytotoxicity assay using LLC-MK2 cell line. protocols.io https://dx.doi.org/10.17504/protocols.io.bjgbkjsn Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: PCR Protocol for Taq DNA Polymerase with ThermoPol® Buffer (M0267)

Proper citation: New England Biolabs 2015. PCR with Taq and ThermoPol® Buffer (M0267). protocols.io https://dx.doi.org/10.17504/protocols.io.cifubm Copy   

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Authors: Lenny Teytelman, protocols.io team, Anita Broellochs
Summary: protocols.io offers extensive flexibility in entering and presenting detailed methods. However, simple suggestions in here can help to make your protocol more useful to you and others. Importantly, easy steps outlined here can greatly increase the visibility of your protocol and ensure that scientists who need can find it.If you have any questions, please feel free to reach out to the protocols.io team by e-mailing us at  info [at] protocols.io. 

Proper citation: Lenny Teytelman, protocols.io team, Anita Broellochs 2019. How to make your protocol more reproducible, discoverable, and user-friendly. protocols.io https://dx.doi.org/10.17504/protocols.io.2yegfte Copy   

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