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Authors: Rex Malmstrom
Group: VERVE Net, Sullivan Lab
Summary: This is a storage solution that stabilizes and protectsRNA while inactivating RNase
Proper citation: Rex Malmstrom 2015. RNAlater Recipe. protocols.io dx.doi.org/10.17504/protocols.io.c56y9d Copy
Authors: Nadia Szeinbaum, Abbigail Johnson, Thomas Swofford
Summary: This protocol describes the step to assemble your own Planetary Domino. We designed this at Georgia Tech for an Astrobiology outreach event called "An Evening of Wonder", which we organized as part of AbGradCon2018. The goal of this activity was to introduce how microorganisms drive many geochemical cycles and how they are interconnected. Each domino piece corresponds to a bacerial functional role/"trophic" role (i.e., aerobic heterotrophs, anaerobic metal reducers, etc.) contains substrates on one side and products on the other. The idea is to find how they can be connected, like pieces of a domino.
Proper citation: Nadia Szeinbaum, Abbigail Johnson, Thomas Swofford 2019. Planetary Dominoes - How microbes drive biogeochemical cycles. protocols.io dx.doi.org/10.17504/protocols.io.8wrhxd6 Copy
Authors: Kiichi Hirota, Yoshiyuki Matsuo
Proper citation: Kiichi Hirota, Yoshiyuki Matsuo 2019. ADP assay (Colorimetric). protocols.io dx.doi.org/10.17504/protocols.io.zk4f4yw Copy
Authors: Joshua Weitz
Group: VERVE Net
Summary: BiMat: a MATLAB package to facilitate the analysis of
bipartite networks.
Extensive documentation, code, and worked examples available on:
http://bimat.github.ioThe datasets included with the package include phage-bacterai infection networks.
Proper citation: Joshua Weitz 2016. BiMat: a MATLAB package to facilitate the analysis of bipartite networks. protocols.io dx.doi.org/10.17504/protocols.io.fajbicn Copy
Authors: Laura Espina
Proper citation: Laura Espina 2020. Taxonomic classification of IonTorrent-sequenced 16S amplicon sequences. protocols.io dx.doi.org/10.17504/protocols.io.bh8pj9vn Copy
Authors: RACHELE CESARONI
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: RACHELE CESARONI 2017. Euplotes crassus transformation using FuGene HD Transfection Reagent as vehicle. protocols.io dx.doi.org/10.17504/protocols.io.g43byyn Copy
Authors: Greg Reeves
Group: OpenPlant Project, Plantae
Summary: The GUS reporter system (GUS: β-glucuronidase) is a reporter gene system, particularly useful in plant molecular biology[1] and microbiology.[2]Several kinds of GUS reporter gene assay are available, depending on the substrate used. The term GUS staining refers to the most common of these, a histochemical technique. (Source: https://en.wikipedia.org/wiki/GUS_reporter_system)
Proper citation: Greg Reeves 2016. GUS Solution. protocols.io dx.doi.org/10.17504/protocols.io.fdibi4e Copy
Authors: Jangsheng Liang
Group: Chemistry Method Development Community
Summary: Potentiometric titrationis can be used to determine the effective content of general chemical . The method offers many advantages, including simplicity, speed, easier end-point determination and more accurate results.
Proper citation: Jangsheng Liang 2018. Potentiometric titration. protocols.io dx.doi.org/10.17504/protocols.io.rkqd4vw Copy
Authors: Francisco Jose Gomez-Garcia, Vicente Vicente Ortega
Summary: When SKH-1 mice are exposed to ultraviolet radiation three times a week for 60min each (a total of 80sessions), with a focus-skin distance of 20 cm, using the sunbed Philips Type HB 554/01/A with 8 Philips Performance S 100W tubes, all animals develope squamous skin carcinomas.
Proper citation: Francisco Jose Gomez-Garcia, Vicente Vicente Ortega 2020. Skin phtocarcinogenesis by sunbed in SKH-1 mice. protocols.io dx.doi.org/10.17504/protocols.io.bc45iyy6 Copy
Authors: Ida Barlow
Group: Behavioural Genomics
Summary: Protocol for screening the disease model C. elegans strains under baseline and bluelight conditions using the Hydra (Loopbio) imaging rigs. This is the protocol for one day of imaging and should be repeated at least three times to get replicates from three separate days. The number of imaging runs depends on the number of strains being screened and whether strains are sorted into 96 well plates row-wise (7 strains per plate) or column-wise (11 strains per plate).
Proper citation: Ida Barlow 2021. Disease model screen protocol. protocols.io dx.doi.org/10.17504/protocols.io.bsicncaw Copy
Authors: Adria D. Lee, Pamela K. Cassiday, Lucia C. Pawloski, Kathleen M. Tatti, Monte D. Martin, Elizabeth C. Briere, M. Lucia Tondella, Stacey W. Martin, The Clinical Validation Study Group
Summary: The appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition. An additional objective was to describe the optimal timing of specimen collection for the various tests. Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit. Patients who had been coughing for ≤ 2 weeks were asked to return in 2-4 weeks for collection of a second, convalescent blood specimen. Sensitivity and specificity of each diagnostic test were estimated using three methods—pertussis culture as the “gold standard,” composite reference standard analysis (CRS), and latent class analysis (LCA). Overall, 868 patients were enrolled and 13.6% were B. pertussis positive by at least one diagnostic test. In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA. CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates. Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough.Timing of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis.
Proper citation: Adria D. Lee, Pamela K. Cassiday, Lucia C. Pawloski, Kathleen M. Tatti, Monte D. Martin, Elizabeth C. Briere, M. Lucia Tondella, Stacey W. Martin, The Clinical Validation Study Group 2018. Standard Operating Procedure for Culture Identification and Differentiation of Bordetella species. protocols.io dx.doi.org/10.17504/protocols.io.kvtcw6n Copy
Authors: Teuta Pilizota, Ekaterina Krasnopeeva, Jerko Rosko
Group: Teuta Pilizota
Summary: The speed of bacterial flagellar motor (BFM) is measured with back-focal-plane interferometry. Heavily attenuated optical trap (855 nm laser) is used to detect the rotation of a polystyrene bead attached to a truncated flagellar filament. Time course of the bead rotation is recorded with the position-sensitive detector.
Proper citation: Teuta Pilizota, Ekaterina Krasnopeeva, Jerko Rosko 2021. BFM speed recording with back-focal-plane interferometry. protocols.io dx.doi.org/10.17504/protocols.io.bch2it8e Copy
Authors: Alemselam Orsango
Summary: SummaryIntroduction: Malnutrition is a public health problem in Ethiopia. Malnutrition can affect children’s cognitive, behavioral, and motor development. Further, healthy development and educational achievement can be compromised due to micronutrient deficiencies including iron deficiency. Different studies show that amaranth grain has better levels of macro and micronutrients especially iron to address the widespread malnutrition problem than other frequently used crops. This useful plant is widely grown in the research area but is considered as a weed rather than a food crop by the community. Furthermore, adequate research has not been conducted in Ethiopia on amaranth’s potential to contribute to reducing the malnutrition problem. This research is initiated to look at amaranth and its potential in reducing iron deficiency anemia among children. The research will inform the public at large, policymakers, and academicians as it deals with an underutilized nutritious crop that has the potential to contribute to reducing the malnutrition problem in the country. Objective: To assess the iron deficiency status of children and to evaluate the effect of amaranth containing bread consumption on the anthropometry and iron deficiency status of children in the age of 24.0-59.9 months. Methodology: A cross-sectional study and a randomized controlled trial will be done. For the survey, random sampling will be used to select 340 children. Hemoglobin, serum ferritin, C- reactive protein, and anthropometric measurements will be taken and used as the baseline data for the experimental study. In the randomized controlled trial, 100 children with a hemoglobin level of less than 11 mg /dl and above 7 mg/dl (mild and moderate anemia) will be recruited into the trial after informed consent. Severely ill individuals will be referred to as Hawassa Referal Hospital. Kebele-stratified simple random sampling will ensure 1:1 allocation to each group. Sequentially numbered, opaque, sealed envelopes will be used to assure concealment of allocation. The study subjects and parents (other caregivers in the absence of parents) will be kept uniformed about the trial allocation. Statistical analysis will be done using the latest SPSS version and ENA for SMART software. The survey will present descriptive statistics including frequencies, means, ranges and demographic characteristics of the population. The trial will be analyzed according to the intention-to-treat principles using logistic crude and site adjusted regression models with a mean difference in Hemoglobin as the primary outcome measure. Conclusion: This research project has the potential to be hypothesis generating and will inform policy makers and scientists about amaranth’s potential to combat malnutrition specifically iron deficiency in the area. Keyword: Amaranth, iron deficiency anemia
Proper citation: Alemselam Orsango 2020. anemia, iron defficency anemia and amaranth trial in Ethiopia. protocols.io dx.doi.org/10.17504/protocols.io.bhzbj72n Copy
Authors: Katy Monteith
Group: Vale Lab
Summary: This protocol describes how to make an egg dechorionator using available lab equipment before describing how to successfully use the egg dechorionator to remove the outer chorion layer from a Drosophila egg. Egg dechorionation allows cellular analysis, quantitative microinjection and the rearing of developing offspring in a gnotobiotic or axenic environment. This protocol is written with the use of dechorionated eggs for rearing fruit flies in a gnotobiotic or axenic environment in mind. Therefore sterile techniques are described. For cellular analysis and/or quantitative microinjection the protocol can be followed but with a less stringent sterile procedure where unneccesary.
Proper citation: Katy Monteith 2018. Drosophila Egg Dechorionation. protocols.io dx.doi.org/10.17504/protocols.io.vcze2x6 Copy
Authors: Caroline Christine Pincela da Costa , Nayane Soares de Lima, Rodrigo da Silva Santos, Angela Adamski da Silva Reis
Group: Molecular Pathology Laboratory
Summary: The VEGF-A gene encodes a homonymous protein, responsible for regulating angiogenesis and vascular permeability. In addition, in the central nervous system, it acts as a neurotrophic factor, stimulating neurogenesis and cell survival. VEGF-A rs28357093 is located in the promoter region of the gene. There are few studies with this polymorphism, with no reports on the consequences. However, it is in a regulatory region, important for the control of gene transcription, and may influence gene and protein expression. In this document, we share our protocol for amplification and enzymatic digestion for this SNP, using the PCR-RFLP technique and silver staining for genotyping analysis.
Proper citation: Caroline Christine Pincela da Costa , Nayane Soares de Lima, Rodrigo da Silva Santos, Angela Adamski da Silva Reis 2021. PCR-RFLP protocols for genotyping VEGF-A rs28357093 . protocols.io dx.doi.org/10.17504/protocols.io.bqvymw7w Copy
Authors: Konstantin J. Bode, Stefanie Mueller, Matthias Schweinlin, Marco Metzger, Thomas Brunner
Group: BioTechniques
Summary: This protocol describes a novel fluorometric method for the quantitative measurement of specific organoid cell death. Organoids are stained simultaneously with the cell impermeable nuclear dye propidium iodide and cell permeable Hoechst 33342. While Hoechst allows in-well normalization to cell numbers, propidium iodide detects relative proportion of dead cells independent of hydrogel. Measurement and analysis time, as well as usability are drastically improved in comparison to other established methods. Parallel multiplexing of this method with established assays measuring mitochondrial activity further enhances its applicability in personalized medicine and drug discovery.
Proper citation: Konstantin J. Bode, Stefanie Mueller, Matthias Schweinlin, Marco Metzger, Thomas Brunner 2019. A fast and simple fluorometric method to detect cell death in 3D intestinal organoids. protocols.io dx.doi.org/10.17504/protocols.io.78vhrw6 Copy
Authors: Jonathan Eisen, Jonathan Eisen Lab
Group: EisenLab, Seagrass Microbiomes
Summary: This collection protocol was developed as part of the Seagrass Microbiome Project. This was originally a collaboration among Jonathan Eisen and Jay Stachowicz at the University of California, Davis and Jessica Green at the University of Oregon, with funding from the Gordon and Betty Moore Foundation.
Proper citation: Jonathan Eisen, Jonathan Eisen Lab 2019. Seagrass Microbiome Sample Collection and Preservation. protocols.io dx.doi.org/10.17504/protocols.io.fxzbpp6 Copy
Authors: James Lyon, Patrick Macdonald, Jocelyn Manning Fox
Group: Human Cell Atlas Method Development Community
Proper citation: James Lyon, Patrick Macdonald, Jocelyn Manning Fox 2019. Human Islet Isolation Media Preparation. protocols.io dx.doi.org/10.17504/protocols.io.sfyebpw Copy
Authors: David Dunigan and Irina Agarkova
Group: VERVE Net
Proper citation: David Dunigan and Irina Agarkova 2016. Dot Blot Preparation. protocols.io dx.doi.org/10.17504/protocols.io.etibeke Copy
Authors: Yulia A. Desheva, Tatiana A. Smolonogina, Ivan A. Sychev
Summary: The NA inhibition test using large molecular fetuin substrate is believed to be the most specific method to detect antibodies against influenza virus neuraminidase. To avoid cross-reacting with hemagglutination-inhibition antibodies we have prepared several reassortant A/H7N1 viruses containing the NA of A/California/07/09(H1N1)pdm, А/New Caledonia/20/1999(H1N1) or A/Vietnam/1203/04(H5N1), and the hemagglutinin derived from A/equine/Prague/1/56(H7N7) influenza virus. Adjustment and validation of the NI test with the reassortant viruses included the determination of the viruses working concentration and selection of optimal conditions for the enzymatic reaction.
Proper citation: Yulia A. Desheva, Tatiana A. Smolonogina, Ivan A. Sychev 2018. Peroxidase-linked lectin assay to determine neuraminidase-inhibiting antibodies using reassortant influenza viruses. protocols.io dx.doi.org/10.17504/protocols.io.n3adgie Copy
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