Protocols   Select Another Resource Report Type

Authors: Benjamin Istace, Anne Friedrich, Léo dAgata, Sébastien Faye, Emilie Payen, Odette Beluche, Claudia Caradec, Sabrina Davidas, Corinne Cruaud, Gianni Liti, Arnaud Lemainque, Stefan Engelen, Patrick Wincker, Joseph Schacherer, Jean-Marc Aury
Group: GigaScience Press
Summary: Describes the library preparation for Nanopore sequencing from low input DNA according to the SQK-MAP006 protocolIt accompanies the GigaScience publication:Benjamin Istace, et al. (2017) De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer. GigaScience...

Proper citation: Benjamin Istace, Anne Friedrich, Léo dAgata, Sébastien Faye, Emilie Payen, Odette Beluche, Claudia Caradec, Sabrina Davidas, Corinne Cruaud, Gianni Liti, Arnaud Lemainque, Stefan Engelen, Patrick Wincker, Joseph Schacherer, Jean-Marc Aury 2017. SQK-MAP006 Low Input protocol for library preparation for Nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.gvwbw7e Copy   

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Authors: avinash.kale
Group: UM-DAE Centre for Excellence in Basic Sciences

Proper citation: avinash.kale 2020. SEM imaging of Bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.bjdjki4n Copy   

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Authors: Alan J. Cone
Summary: Separates molecules based on size. Great for checking DNA after a Restriction Digest.

Proper citation: Alan J. Cone 2016. Gel Electrophoresis. protocols.io https://dx.doi.org/10.17504/protocols.io.ey3bfyn Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the general procedures used for intramuscular injection in adult mice. Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Proper citation: Allen Institute for Brain Science 2020. Intramuscular Injection Adult Mouse. protocols.io https://dx.doi.org/10.17504/protocols.io.bfzfjp3n Copy   

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Authors: Li Deng & Jennifer Brum
Group: VERVE Net, Sullivan Lab

Proper citation: Li Deng & Jennifer Brum 2016. SYBR Gold Staining for viral enumeration using 13 mm Anodisc 0.02 ?m filters. protocols.io https://dx.doi.org/10.17504/protocols.io.c7cziv Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Hepatic gluconeogensis is estimated using pyruvate tolerance test that measures systemic elevation of glucose partly derived from pyruvate and hepatic gluconeogensis following an intraperitoneal bolus injection of pyruvate in awake mice. Hepatic gluconeogenesis is affected by obesity and regulates glucose homeostasis.

Proper citation: Jason Kim 2019. U Mass - Hepatic gluconeogenesis. protocols.io https://dx.doi.org/10.17504/protocols.io.xy7fpzn Copy   

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Authors: Demian F Gomez, Andrew J. Johnson
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol explains the process of taking high quality photos of bark and ambrosia beetles, creating the image stacks, and processing the images in image editing software (Gimp or Photoshop).This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.

Proper citation: Demian F Gomez, Andrew J. Johnson 2020. Ultra-High Macro Photography of Bark Beetles. protocols.io https://dx.doi.org/10.17504/protocols.io.bnvgme3w Copy   

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Authors: Qiaoshan Lin

Proper citation: Qiaoshan Lin 2017. RNA-seq analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.k7vczn6 Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: The lipid extraction method is based on work by Folch 1957. Tissue is homogenized and put into a 2:1 chloroform and methanol mix. The mix is separated into two phases by adding a salt solution. The upper phase contains any non-lipid substances and the lower phase contains the chloroform with the lipids. The upper phase is removed and a sample is taken from the bottom phase and evaporated. The sample is reconstituted with 2-propanol with a volume to bring it to the desired concentration to assay. Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3- phosphate and adenosine diphosphate. Glycerol-3-phosphate is oxidized by dihydroxyacetone phosphate (DAP) by glycerolphosphate oxidase producing hydrogen peroxide (H²O²). In a Trinder5 type color reaction catalyzed by peroxidase, the H²O² reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2- hydroxybenzene sulfonate (DHBS) to produce a red colored dye. The absorbance of this dye is proportional to the concentration of triglycerides present in the sample. Cholesterol esters are enzymatically hydrolysed by cholesterol esterase to cholesterol and free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholest-4-en-3- one and hydrogen peroxide. The hydrogen peroxide combines with HBA and 4-aminoantipyrine to form a chromophore (quinoneimine dye) which may be quantitated at 500-550nm.

Proper citation: Peter Havel 2019. UC Davis - Tissue TG & TC Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.62thgen Copy   

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Authors: Remco Stam
Group: Stam Lab
Summary: Growth medium used at the Technical University Munich, Phytopathology, for cultivation of Fusarium spp.Note, we use 1/4 concentration.

Proper citation: Remco Stam 2016. 0.25 Potato Dextrose Agar. protocols.io https://dx.doi.org/10.17504/protocols.io.fmnbk5e Copy   

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Authors: Oscar Debrah, Ernest Mensah, Laura Senyonjo, Dziedzom K. de Souza, Tei E. Hervie, David Agyemang, Didier Bakajika, Benjamin Marfo, Felix Ahorsu, Seth Wanye, Joseph Koroma, Agatha Aboe, Nana-Kwadwo Biritwum
Summary: Background: In order to achieve elimination of blinding trachoma, a country needs to demonstrate that the elimination prevalence thresholds have been surpassed and then sustained for a three year period. Ghana achieved the thresholds in 2008, and since 2011 have been implementing their trachoma surveillance strategy, which includes community and school screening for signs of follicular trachoma and trichiasis, in each trachoma-endemic district. In 2015, the country plans to conduct a number of district level population based survey to verify elimination of blinding trachoma. This decision is currently made based on prevalence of clinical evaluation of “trachomatous trichiasis” and “trachomatous inflammation – follicular” (TF), the second of which has been shown to be a problematic indicator in low prevalence settings. Further evidence is required to determine the optimal surveillance strategy and indicators for trachoma.Objectives: This study will determine if Ghana has sustained the elimination prevalence thresholds. Additionally, the study will review the trachoma surveillance strategy employed in Ghana and if it was able to identify any potential resurgence of infection. It will also assess the relationships between the prevalence of the clinical sign TF, compared to prevalence of infection and antibody levels, after completion of what most observers believe to have been a successful programme. This will help to determine whether it may be appropriate to consider one or more alternative indicators for validating elimination of blinding trachoma. Surveillance for other infections (including yaws and other NTDs) will also be integrated into the pre-validation trachoma survey.Methods: The standard World Health Organization (WHO)-recommended population-based prevalence survey will be conducted in areas already scheduled to conduct validation surveys in 2015, but with the addition of eye swabbing to test for infection and finger pricking to collect bloodspots for antibody testing. Villages identified with a TF prevalence ≥5%, either retrospectively, during the on-going surveillance or during the district level survey (index village), will result in focal screening of children in neighbouring villages (and the ‘index’ village), including a clinical assessment and tests for trachoma infection and antibody levels. Surveillance for additional infections will also be included through antibody testing using the same sera collected for trachoma. Finally, document analysis and qualitative methodologies will be employed to review the operationalization of the trachoma surveillance system in Ghana.Impact: Results from this study will provide the Ghana Health Service with evidence, as to whether the country has sustained their elimination targets for trachoma. It will also inform the WHO-led Alliance for the Global Elimination of Trachoma by 2020 (GET2020) on the appropriateness of alternative indicators and surveillance methodologies for verification of blinding trachoma.

Proper citation: Oscar Debrah, Ernest Mensah, Laura Senyonjo, Dziedzom K. de Souza, Tei E. Hervie, David Agyemang, Didier Bakajika, Benjamin Marfo, Felix Ahorsu, Seth Wanye, Joseph Koroma, Agatha Aboe, Nana-Kwadwo Biritwum 2017. Pre-validation survey for the elimination of trachoma and evaluation of the effectiveness of the trachoma surveillance strategy in Ghana. protocols.io https://dx.doi.org/10.17504/protocols.io.h9db926 Copy   

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Authors: Susan A. Kimmance and Corina P. D. Brussaard
Group: VERVE Net
Summary: The modified dilution assay aims to partition phytoplankton mortality into virus- versus grazing-induced fractions and has previously been applied to several different environments to determine viral lysis rates of natural phytoplankton. The method involves creating a gradient of both grazing and viral lysis by dilution with different proportions of grazer- and virus-free filtrate, and assessing the subsequent impact on phytoplankton growth rates.Kimmance, S. A., and C. P. D. Brussaard. 2010. Estimation of viralinduced phytoplankton mortality using the modified dilution method, p. 65–73. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.

Proper citation: Susan A. Kimmance and Corina P. D. Brussaard 2016. Estimation of viral-induced phytoplankton mortality using the modified dilution method. protocols.io https://dx.doi.org/10.17504/protocols.io.dpf5jm Copy   

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Authors: HANNIGAN GD, GRICE EA, ET AL.
Group: VERVE Net, Club Grice
Summary: This protocol outlines the analysis used to plot MEGAN taxonomic assignments. Based on the methods from the following publication:Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." mBio 6.5 (2015): e01578-15.

Proper citation: HANNIGAN GD, GRICE EA, ET AL. 2016. Script R9: Plotting Microbial Taxonomy from MEGAN. protocols.io https://dx.doi.org/10.17504/protocols.io.ejdbci6 Copy   

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Authors: Qi Wang, Qiang Sun, Xiaoping Li, Zhefeng Wang, Haotian Zheng, Yanmei Ju, Ruijin Guo, Songlin Peng, Huijue Jia
Group: BGI, GIGA, GigaScience Press
Summary: Bone mass loss contributes to the risk of bone fracture in the elderly. Many factors including age, obesity, estrogen and diet, are associated with bone mass loss. Mice studies suggested that the gut microbiome might affect the bone mass by regulating the immune system, however there has been little evidence from human studies. Bone loss increases after menopause. Therefore, we have recruited 361 Chinese post-menopausal women to collect their fecal samples and metadata to conduct metagenome-wide association study (MWAS) to investigate the influence of the gut microbiome on bone health. Gut microbiome sequencing data were produced using BGISEQ500 sequencing, Bone mineral density (BMD) was calculated using Hologic dual energy X-ray machine, body mass index (BMI) and age were also recorded.This collected data allows exploration of the gut microbial diversity and their links to bone mass loss, as well as microbial markers for bone mineral density. In addition, these data are potentially useful in studying the role the gut microbiota might play in bone mass loss and in exploring the bone mass loss process.

Proper citation: Qi Wang, Qiang Sun, Xiaoping Li, Zhefeng Wang, Haotian Zheng, Yanmei Ju, Ruijin Guo, Songlin Peng, Huijue Jia 2021. Protocols for "Shotgun Metagenomics of 361 elderly women reveals gut microbiome change in bone mass loss". protocols.io https://dx.doi.org/10.17504/protocols.io.bq6hmzb6 Copy   

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Authors: Johnny O Farnen
Group: Bellevue University Natural Sciences Laboratories
Summary: Loess (decayed limestone) topsoil based soil and water media concentrate for use with RO/DI water. NOT for use with treated tap or spring water! Intended for use in filamentous algae, volvox and euglenoid culture.

Proper citation: Johnny O Farnen 2018. Loess Soil-Water Medium Concentrate (Stock Solution) for use with RO/DI ONLY. protocols.io https://dx.doi.org/10.17504/protocols.io.nxmdfk6 Copy   

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Authors: Girija Goyal
Summary: Adherent cells were cultured in flat bottom plates and fixed in situ. This is a protocol to detect specific proteins by immunofluorescence in these cells. Immunofluoroscence is a common laboratory technique where specific proteins within cells can be detected using antibodies coupled to fluorophores and then visualized using a microscope. The cells were counterstained with Hoechst dye to label nuclei.

Proper citation: Girija Goyal 2019. Basic immunofluorescence protocol for adherent cells. protocols.io https://dx.doi.org/10.17504/protocols.io.wt4feqw Copy   

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Authors: Caroline Storer, Jiri Hulcr
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol describes how to collect and preserve the red turpentine beetle (RTB), Dendroctonus valens.This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.

Proper citation: Caroline Storer, Jiri Hulcr 2020. Collecting the Red Turpentine Beetle. protocols.io https://dx.doi.org/10.17504/protocols.io.bnummeu6 Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Anti-Neu5Gc may be used for staining cells prior to analysis by Flow Cytometry. This kit contains all the essential components needed to identify Neu5Gc on the surface of cells by flow cytometry.Use of the blocking agent (Neu5Gc Assay Blocking Solution) provided in the kit is essential, as commonly used blocking agents invariably contain serum, or serum components, that can either inhibit detection or introduce Neu5Gc contamination.Tissue culture-grown CHO-K1 cells can be used as a positive control, and human peripheral blood mononuclear cells serve as a negative control. Adherent tissue culture-grown cells should be released from the culture flasks by using 5-10mM EDTA for 10 minutes at room temperature. Other non-enzymatic methods, such as Accutase®, may be used. Immediately wash cells in blocking buffer that contains a lower concentration of EDTA, and resuspend cells in blocking buffer to determine cell numbers and viability.It is assumed that the user is familiar with the general principles and practices of Flow Cytometry.

Proper citation: Sam Li 2019. Anti-Neu5Gc Antibody Kit Protocol - Flow Cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.baaqiadw Copy   

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Authors: Peng Wang, Hongjian Ni, Ruihe Wang

Proper citation: Peng Wang, Hongjian Ni, Ruihe Wang 2018. Fig 7. Effect of amplitude and frequency of pulse pressure on weight on bit. A. Frequency of pulse pressure f = 24Hz. B. Amplitude of pulse pressure P0 =2MPa. protocols.io https://dx.doi.org/10.17504/protocols.io.ke8cthw Copy   

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Authors: Michael Van Dyke, James Cox
Summary: Protein-DNA binding interactions are critical in several biological processes, especially the regulation of gene expression at the level of transcription initiation. An important technique for studying these interactions is the electrophoretic mobility shift assay (EMSA), whereby protein-DNA complexes are resolved on the basis of their mass:charge ratio using native polyacrylamide gel electrophoresis (nPAGE). Here we describe EMSA using PCR-generated, near infrared-fluorescent DNA probes, and IR fluorescence imaging to qualitatively and quantitatively study the interaction of transcriptional regulatory proteins from thermophilic organisms with different DNAs. Direct imaging of IR fluorophore-labeled DNA probes is advantageous because it provides high sensitivity (subnanomolar) without the need for intermediate staining steps or costly and problematic radiolabeled probes, thereby providing a more affordable and sensitive option to image protein-DNA on polyacrylamide gels by techniques such as EMSA.

Proper citation: Michael Van Dyke, James Cox 2018. Electrophoretic Mobility Shift Assays using Infrared-Fluorescent DNA Probes. protocols.io https://dx.doi.org/10.17504/protocols.io.mbdc2i6 Copy   

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