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Authors: Prince Samoh

Proper citation: Prince Samoh 2020. Protocol for Preparing SOC. protocols.io https:// Copy   

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Authors: Anita Iyer
Group: Coronavirus Method Development Community

Proper citation: Anita Iyer 2020. SARS-CoV-2 RBD ELISA. protocols.io https:// Copy   

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Authors: J. Greg Caporaso, Gail Ackermann, Amy Apprill, Markus Bauer, Donna Berg-Lyons, Jason Betley, Noah Fierer, Louise Fraser, Jed A. Fuhrman, Jack A. Gilbert, Niall Gormley, Greg Humphrey, James Huntley, Janet K. Jansson, Rob Knight, Chris L. Lauber, Catherine A. Lozupone, Sean McNally, David M. Needham, Sarah M. Owens, Alma E. Parada, Rachel Parsons, Geoff Smith, Luke R. Thompson, Luke Thompson, Peter J. Turnbaugh, William A. Walters, Laura Weber
Group: Earth Microbiome Project
Summary: The 16S protocol detailed here is designed to amplify prokaryotes (bacteria and archaea) using paired-end 16S community sequencing on the Illumina platform. Primers 515F-806R target the V4 region of the 16S SSU rRNA.For running these libraries on the MiSeq and HiSeq, please make sure you read the supplementary methods of Caporaso et al. (2012). You will need to make your sample more complex by adding 5-10% PhiX to your run.

Proper citation: J. Greg Caporaso, Gail Ackermann, Amy Apprill, Markus Bauer, Donna Berg-Lyons, Jason Betley, Noah Fierer, Louise Fraser, Jed A. Fuhrman, Jack A. Gilbert, Niall Gormley, Greg Humphrey, James Huntley, Janet K. Jansson, Rob Knight, Chris L. Lauber, Catherine A. Lozupone, Sean McNally, David M. Needham, Sarah M. Owens, Alma E. Parada, Rachel Parsons, Geoff Smith, Luke R. Thompson, Luke Thompson, Peter J. Turnbaugh, William A. Walters, Laura Weber 2018. EMP 16S Illumina Amplicon Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.nuudeww Copy   

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Authors: Hansheng Zhao

Proper citation: Hansheng Zhao 2018. Genome-wide identification of genes involved in the lignin biosynthetic pathway. protocols.io https://dx.doi.org/10.17504/protocols.io.phjdj4n Copy   

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Authors: Cristina Corral
Group: George Lab

Proper citation: Cristina Corral 2020. ANY-maze Protocol (Elevated Plus Maze) v6.2. protocols.io https:// Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the process to obtain electrophysiological recordings and cellular contents from neurons in postnatal mouse and/or human brain slices.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. Patch-Seq Recording and Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bepyjdpw Copy   

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Authors: Magdalena Julkowska
Group: Salt Lab KAUST
Summary: Protocol adapted from Shin et al., 2005 and Orman-Ligeza et al. 2016 (citing Shin)

Proper citation: Magdalena Julkowska 2020. ROS staining. protocols.io https://dx.doi.org/10.17504/protocols.io.peidjce Copy   

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Authors: Gwendolyn Gallagher
Group: Coleman Lab
Summary: Purpose: Preparation of culture samples for mass spectrometry-based proteomicsPrinciple: Utilizing a membrane-enrichment method of lysing cells and preparing peptides has yielded higher representation of membrane proteins in our mass spectrometry-based proteomic results.Traditional methods do not adequately extract or digest hydrophobic, transmembrane proteins. Particularly, we can now see full expression patterns of proteorhodopsin, something we could not detect using traditional mass spec proteomics prep.This protocol builds on the work of Molloy (2008) Methods Mol Biol (doi:10.1007/978-1-60327-064-9_30), Erde et al. (2014) J. Proteome Res. (doi:10.1021/pr4010019), and Waldbauer, et al. (2017) Anal. Chem. (doi: 10.1021/acs.analchem.7b02752).Pure culture samples were spun down and flash frozen for proteomics.A carbonate extraction protocol was used for membrane enrichment before eFASP.The membrane fraction was enzymatically digested with both chymotrypsin and trypsin and the cytosolic fraction was digested with just trypsin.These samples were then ready to be processed further by in vitroisotopic peptide labeling (diDO-IPTL).

Proper citation: Gwendolyn Gallagher 2020. A membrane-enriched preparation of culture samples for mass spectrometry-based proteomics. protocols.io https://dx.doi.org/10.17504/protocols.io.bdapi2dn Copy   

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Authors: Elizabeth Jannaman, M. Sofia Ortega, Tad S. Sonstegard, Peter Hansen
Group: University of Florida Bovine Embryo Lab
Summary: Several mutations exist in the prolactin receptor gene (PRLR) of cattle that cause a short, sleek haircoat.. The mutation is dominant so slick-haired cattle can have either one ortwo copies of the mutation. The assay described here detects the most-well studied mutation (SLICK1)that causes translation of a truncated PRLR of 461 amino acids. Originally described in Senepol cattle,this mutation has also been introduced into Holsteins in Puerto Rico, Florida and elsewhere. Note thatthe assay will not detect other mutations in PRLR that also cause the slick phenotype.

Proper citation: Elizabeth Jannaman, M. Sofia Ortega, Tad S. Sonstegard, Peter Hansen 2019. PCR-based assay for genotyping of the slick mutation in cattle. protocols.io https://dx.doi.org/10.17504/protocols.io.6kxhcxn Copy   

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Authors: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: Protocol for collection, histological processing, and imaging of rat vagus nerves.

Proper citation: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill 2019. SPARC_Duke_Grill_OT2-OD025340_RatVagusNerveCollectionHistologyMicroscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.ww3ffgn Copy   

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Authors: Rebecca Maher

Proper citation: Rebecca Maher 2018. Instructions for Planar Area Analysis using CPCe 4.1 Software. protocols.io https://dx.doi.org/10.17504/protocols.io.pmvdk66 Copy   

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Authors: Michiyo Tsuru
Group: Clinical Proteomics and Gene Therapy Laboratory
Summary: We generated a miR-340 probe by tagging Has-miR-340 aatcaG(L)t5(L)aT(L)tG(L)cT(L)ttataa_N(6)_Y with Alexa Fluor 488. LNA-in situ hybridization (ISH) was performed according to the manufacturer’s instructions (http://www.exiqon.com/mirna-ish-kit). Sections of FFPE tissues of human OPLL and null mice (4-µm-thick) were fixed with 4 % PFA in PBS for 20 min at room temperature, washed with PBS (3 × 5 min), treated with 0.5 % Triton X-100 (10 min at 4 °С), and briefly washed with PBS followed by two washes (10 min each) in saline-sodium citrate buffer (2× SSC, 0.3 M NaCl, 0.03 M Na3С6Н5О7, рН 7.0). The sections were then digested with 15 μg/mL proteinase K (Exiqon, Vedbaek, Denmark) at 37 °C for 10 min and rinsed for 3 × 5 min in PBS. Hybridization was performed in a humid chamber for 18 h at 65 °C. In situ hybridization of miR-340 was conducted using a miRCURY LNA microRNA ISH Optimization kit (Exiqon, Vedbaek, Denmark). miR-340-Alexa Fluor 488 and bone morphogenetic protein 2 (Alexa Fluor 555) were detected in human OPLL tissue by in situ hybridization and immunocytochemistry, respectively. Nuclei were counterstained with DAPI. After staining, the tissues were observed by fluorescence microscopy (BZ-X700, Keyence, Osaka, Japan).

Proper citation: Michiyo Tsuru 2018. In situ hybridization. protocols.io https://dx.doi.org/10.17504/protocols.io.j9hcr36 Copy   

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Authors: Devang Mehta

Proper citation: Devang Mehta 2017. Cassava leaf nucleic acid extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.iavcae6 Copy   

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Authors: Kenji Ohgane
Summary: Sometimes it is desirable to fit sigmoid curves with shared parameters against several data sets. Such simultaneous fitting procedure is available in most of the specialized statistical softwares (R, Prism etc.), but also possible with Excel. Here, this protocol explains how to perform simultaneous fitting using Excel Solver plug-in against a pair of data set. For more basic protocol to fit sigmoid curve, see Kenji Ohgane (2019) Sigmoid fitting in Excel (Excel Solver Add-In). protocols.io dx.doi.org/10.17504/protocols.io.78ihrue. See Gerdi Kemmer & Sandro Keller (2010) Nat. Protocols 5: 267–281 for more detail on the use of Excel Solver in nonlinear least square fitting.

Proper citation: Kenji Ohgane 2019. Simultaneous fitting of sigmoid curves in Excel (Excel Solver Add-in). protocols.io https://dx.doi.org/10.17504/protocols.io.8g9htz6 Copy   

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Authors: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen
Group: VERVE Net, Suttle Laboratory of Marine Molecular Microbiology and Virology
Summary: More than 40 years ago, Safferman and Morris (1963) used the plaque assay method to isolate the first cyanophage that infects a freshwater filamentous cyanobacterium, Plectonema boryanum. Since then, this approach has been used successfully to detect and isolate a number of different phages infecting marine Synechoccocus and Prochlorococcus (e.g., Suttle and Chan 1993; Waterbury and Valois 1993; Wilson et al. 1993; Sullivan et al. 2003).The advantages of this method are that results are easily interpreted as plaques formed on pigmented lawns can be easily identified. Since a plaque is the result of a single infection event, the virus can be easily purified and cloned. The first part of the procedures described as follows: (1) preparation of base plates and (2) preparation of top agar/agarose are applicable also for bacteriophage plaque assays.

Proper citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. Isolation of cyanophages by plaque assays. protocols.io https://dx.doi.org/10.17504/protocols.io.dqg5tv Copy   

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Authors: Timothy Nichols, David Clemmons
Group: Diabetic Complications Consortium
Summary: This assay is used by the DiaComp to measure glucose tolerance and insulin sensitivity in pigs.Diabetic Complications:Reference:1. Bergman RN, Finegood DT, Ader M: Assessment of insulin sensitivity in vivo. Endocr Rev 1985, 6:45-86.

Proper citation: Timothy Nichols, David Clemmons 2019. Frequently sampled Insulin glucose tolerance test. protocols.io https://dx.doi.org/10.17504/protocols.io.8e8hthw Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Phenotyping of Rodents for the Presence of Diabetic Neuropathy In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.Diabetic Complication:Reference: AFIP Laboratory Methods in Histotechnology, 1994, p. 16, 53-55.

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Hematoxylin and Eosin Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.3jsgkne Copy   

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Authors: Jing Li

Proper citation: Jing Li 2018. Soil microbial community structure -PLFAs. protocols.io https://dx.doi.org/10.17504/protocols.io.nggdbtw Copy   

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Authors: Justin Ryan, Nicole Coufal, Sage Aronson, Kelsey Ladt, Catelyn Andersen, Mark Zeller, Stephen Rawlings, Denise Malicki, Gene Yeo
Group: Coronavirus Method Development Community
Summary: The global pandemic due to SARS-CoV2 virus, also known as COVID-19, has drastically increased the need for nasopharyngeal-swab-based testing resulting in shortages of commercially available nasopharyngeal (NP) swabs. One solution to overcome the national deficit of swabs is for medical device manufacturers and hospitals to generate NP swabs. Numerous entities are attempting to manufacture a direct from 3D printing NP swab but presented here is a validated two-part swab manufacturing protocol utilizing 3D printing and manual intervention (wrapping of nylon fibers). We recommend material extrusion or powder bed fusion technologies utilizing materials that can be sterilized using high level heat decontamination. Through the application of 3D printing and manual fabrication, we present an NP swab that can be created in a controlled environment. Coupled with CDC published viral transport media, the following swab can be used for COVID-19 testing or for testing for other respiratory viruses (eg, influenza, respiratory syncytial virus). The CDC viral transport medium has only four reagents which are readily available. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Justin Ryan, Nicole Coufal, Sage Aronson, Kelsey Ladt, Catelyn Andersen, Mark Zeller, Stephen Rawlings, Denise Malicki, Gene Yeo 2020. 3D Printed Nasopharyngeal Swabs with Wrapped Rayon Fibers Developed and validated by SCREEN (San Diego Covid19 Research Enterprise Network). protocols.io https://dx.doi.org/10.17504/protocols.io.bemxjc7n Copy   

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Authors: Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Daniel Richter 2017. Modified Qiagen PCR purification (no gel extraction) with MinElute spin column.. protocols.io https://dx.doi.org/10.17504/protocols.io.ivece3e Copy   

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