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Authors: Kent Lloyd
Group: Mouse Metabolic Phenotyping Centers
Summary: This is a generic protocol for making genetically-altered mouse models for research. Investigators who do not have a mutant mouse to send for testing at the MMPC @ UC Davis can ask for the mutant mouse to be derived by the MMPC @ UC Davis, through the UC Davis Mouse Biology Program. Genetically altered mice can be made on most genetic backgrounds, such as C57BL/6, FVB/N, B6D2F1 and other hybrids, and others. Transgenic (random genomic integration of expression-competent transgenes) and gene targeted (specific insertion and/or replacement of an endogenous genetic locus) mutant mice can generated. “Knockout” mice can be made so as to delete endogenous gene expression ubiquitously and conditionally in either a specific cell or tissue type (spatial conditional) and/or at a specific age (temporal conditional). Numerous technologies are available for the derivation of mutant mice, including molecular construct design, gene targeted in embryonic stem (ES) cells, pronuclear microinjection of transgenic constructs into zygotes, ES cell microinjection into blastocysts, genome editing in ES cells and zygotes (e.g., TALENs, CRISPR/Cas9). Mutant mice can be genotype confirmed (see MMPC item D2004) and custom bred to generate cohorts of male and/or female mice for phenotyping at the MMPC @ UC Davis.

Proper citation: Kent Lloyd 2019. UC Davis - Mouse Model Creation. protocols.io https://dx.doi.org/10.17504/protocols.io.56gg9bw Copy   

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Authors: Tinka Beemsterboer, Lukas Snoek, H.steven Scholte
Group: Spinoza Centre, REC-L
Summary: Dear researcher,This protocol gives you the steps to start an MRI project at the Spinoza Centre Roeterseiland. If you haven't had contact with Steven Scholte or Tinka Beemsterboer, make sure to contact them before completing this protocol. You can reach us by email:Steven Scholte: [email protected] Beemsterboer: [email protected] you have any suggestions for improving the workflow, don't hesitate to add comments to the steps in the protocol.Hope to see you soon!The Spinoza REC team,Steven Scholte, Tinka Beemsterboer, and Lukas Snoek 

Proper citation: Tinka Beemsterboer, Lukas Snoek, H.steven Scholte 2018. Before data acquisition -- Start MRI Project Spinoza REC. protocols.io https://dx.doi.org/10.17504/protocols.io.pdxdi7n Copy   

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Authors: Shripal Gandhi, Santo Purnama, Keyur Patel, Praveen Sukumara, Dr Rinu R Ravi
Group: Coronavirus Method Development Community, SensingSelfMission, XPRIZE Rapid Covid Testing
Summary: Summary:The S4 Multiplex Covid-19 /MERS-CoV/ Influenza A/B Rapid Antigens Test Kit developed by Sensing Self Pte. Ltd. can quickly and qualitatively detect Novel Coronavirus / Influenza A/ Influenza B /MERS in human saliva/sputum/stool samples. It can be used as an aid for COVID-19 diagnosis. It provides an aid in the diagnosis of infection by the above viruses.Objective:According to the clinical trial plan, the S4 Multiplex Covid-19 /MERS-CoV/ Influenza A/B Rapid Antigen test kit or “test reagent”, is to test saliva/sputum/stool samples from healthy subjects and confirmed patients with infections caused by these viruses. Test results are compared with another commercial Covid-19 /MERS-CoV/ Influenza A/B nucleic acid detection kit, which is defined as the “gold standard”. The sensitivity, specificity, and total agreement rate are used to evaluate the feasibility of the test reagent in clinical applications.Method:A collection of clinical samples were examined by the S4 Multplex Covid-19 /MERS-CoV/ Influenza A/B Rapid Antigen test kit and the gold standard SARS-COV-2/MERS-CoV/ Influenza A/B nucleic acid detection kit in parallel, to calculate the clinical sensitivity, clinical specificity and total agreement rate of the test reagent.Results:For SARS-CoV-2, compared to the gold standard:In Saliva, the clinical sensitivity of test reagent reached 88.3%, the clinical specificity reached 100.0%In Sputum, the clinical sensitivity of test reagent reached 93.3%, the clinical specificity reached 100.0% In Stool, the clinical sensitivity of test reagent reached 93.3%, the clinical specificity reached 100.0%For MERS, compared to the gold standard:In Saliva, the clinical sensitivity of test reagent reached 86%, the clinical specificity reached 100.0%In Sputum, the clinical sensitivity of test reagent reached 90%, the clinical specificity reached 100.0% In Stool, the clinical sensitivity of test reagent reached 90%, the clinical specificity reached 100.0%For Influenza Virus A, compared to the gold standard:In Saliva, the clinical sensitivity of test reagent reached 85.7%, the clinical specificity reached 98.3%In Sputum, the clinical sensitivity of test reagent reached 91.4%, the clinical specificity reached 100.0% In Stool, the clinical sensitivity of test reagent reached 90%, the clinical specificity reached 100.0%For Influenza Virus B, compared to the gold standard:In Saliva, the clinical sensitivity of test reagent reached 82.9%, the clinical specificity reached 100.0%In Sputum, the clinical sensitivity of test reagent reached 87.1%, the clinical specificity reached 96.7% In Stool, the clinical sensitivity of test reagent reached 85.7%, the clinical specificity reached 98.3%Conclusions:The performance of test reagent has a high agreement rate with the gold standard, proving its good feasibility in diagnosing suspected Covid-19 /MERS-CoV/ Influenza A/B cases.

Proper citation: Shripal Gandhi, Santo Purnama, Keyur Patel, Praveen Sukumara, Dr Rinu R Ravi 2020. SensingSelf S4 Multiplex Covid-19 /MERS-CoV/ Influenza A/B Rapid Antigens Test Kit (Saliva/Sputum/Stool). protocols.io https://dx.doi.org/10.17504/protocols.io.bkwckxaw Copy   

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Authors: Priota Islam
Group: Behavioural Genomics
Summary: Bacterial glycerol stocks are important for long-term storage. Bacteria on LB agar plate can be stored at 4°C for a few weeks. However, if you want to store bacteria for a longer time, you will need to establish glycerol stocks. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years.

Proper citation: Priota Islam 2020. Creating Bacterial Glycerol Stocks for Long-term Storage. protocols.io https://dx.doi.org/10.17504/protocols.io.bf5qjq5w Copy   

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Authors: Grainne O´Brien; Aleš Tichý, Ales Tichy
Summary: Gene expression - MQRT-PCRReverse transcriptase reactions were performed using High Capacity cDNA Reverse transcription kit (Applied Biosystems, FosterCity, CA, USA) according to the manufacturer ’ s protocol with 350 ng of total RNA. Multiplex quantitative RT-PCR Real-time PCR was performed using Rotor-Gene Q (Qiagen, Hilden, Germany). All reactions were run in triplicate using PerfeCTa® MultiPlex qPCR SuperMix (Quanta Biosciences, Inc. Gaithersburg, MD, USA) with primer and probe sets for target genes at 300 nM concentration each. 3 ′ 6-Carboxyfluorescein (FAM), 6-Hexachlorofluorescein (HEX), Texas Red and CY5 (all from Eurogentec Ltd, Fawley, Hampshire, UK) were used as fluorochrome reporters for the hydrolysis probes analysed in multiplexed reactions with between 2 and 4 genes per run including the control. Cycling parameters were 2 min at 95°C, then 45 cycles of 10 s at 95°C and 60 s at 60°C. Data were collected and analyzed by Rotor-Gene Q Series Software. Gene target Ct (cycle threshold) values were normalized to a Hypoxanthine-Guanine phosphoribosyltransferase 1 (HPRT1) internal control. Ct values were converted to transcript quantity using standard curves obtained by serial dilution of PCR-amplified DNA fragments of each gene. The linear dynamic range of the standard curves covering six orders of magnitude (serial dilution from 3.2 x 10-4 to 8.2 x 10-10) gave PCR efficiencies between 93% and 103% for each gene with R2 > 0.998. Relative gene expression levels after irradiation were determined relative to unexposed controls.

Proper citation: Grainne O´Brien; Aleš Tichý, Ales Tichy 2018. MQRT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.mjmc4k6 Copy   

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Authors: Ludimila Labanca, Júlia Fonseca de Morais Caporali, Maurício Campelo Tavares, Denise Utsch Gonçalves
Summary: Galvanic vestibular stimulation (GVS) is a method that stimulates the vestibular afferents with a small current, triggering a motor reflex response in the posture-controlling muscles. This response can be captured by electromyography (EMG) and is called vestibular evoked myogenic potential (VEMP). The VEMP triggered by GVS (galvanic-VEMP) assesses the central pathways of the vestibular system. When the EMG responses are recorded in the lower limbs, galvanic-VEMP provides information about the function of the vestibulospinal tract extending from the cervical to the lumbar spine. Galvanic-VEMP is a safe, low-cost, and easily performed test which has been used to investigate spinal cord function in cases of trauma, tumor, ischemia, and infection. Our research group has been using galvanic-VEMP to follow patients with diseases that cause postural disabilities, such as Human T-cell lymphotropic virus type 1-associated myelopathy (HAM), Schistosomal myeloradiculopathy, and Parkinson’s disease. This protocol aims at showing how to perform VEMP triggered by galvanic vestibular stimulation with the EMG responses being recorded in the lower limbs.

Proper citation: Ludimila Labanca, Júlia Fonseca de Morais Caporali, Maurício Campelo Tavares, Denise Utsch Gonçalves 2018. Vestibular-evoked myogenic potential (VEMP) triggered by galvanic vestibular stimulation recorded in the lower limbs. protocols.io https://dx.doi.org/10.17504/protocols.io.nxbdfin 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.Here, we describe an illuminated orbital shaker designed for culture of microalgae suspensions. It was optimized for production cost, simplicity, low power consumption, design flexibility, and consistent and controllable growth light intensity. The instrument agitates and illuminates microalgae suspensions grown inside flasks. The agitation speed is variable while the light intensity is both variable and programmable (24-hour light/dark diurnal cycle).The instrument offers a vastly cheaper alternative to commercial instruments for many laboratory applications, making it especially well suited for teaching and poorly-resourced research laboratories. It improves on home-built microalgae growth systems by offering consistent and well characterized illumination light intensity, low power consumption and heat dissipation. The illuminated growth area is 20 cm × 15 cm, which is suitable for three T75 tissue culture flasks or six 100 ml Erlenmeyer flasks. The photosynthetic photon flux density, measured inside an Erlenmeyer flask, is variable in eight steps (26−800 μmol·m−2·s−1). The overall material cost is around £300 (including an entry-level orbital shaker). The build takes about two days, requiring electronics assembly and machine workshop skills. The instrument build is documented in a set of protocols, design files, and source code. Its design can be readily modified, scaled, and adapted for other orbital shakers and specific experimental requirements.The illuminated orbital shaker is ideal for growing small volumes of microalgae for research and teaching. It can readily replace commercial systems in many common microalgae culture application for a fraction of their cost. It outperforms typical home-made systems by offering consistent and predictable illumination light intensity and low power consumption.

Proper citation: Jakub Nedbal 2020. Illuminated Orbital Shaker for Microalgae Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.bdtyi6pw Copy   

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Authors: Nus Igem

Proper citation: Nus Igem 2019. Hi-Fi Gibson Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.8abhsan Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. Macrophages mostly make IL-18, which can be produced by hepatocytes and keratinocytes. Its principal target is a co-factor in Th1 cell induction. It causes interferon gamma production and enhances NK cell activity. [1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.

Proper citation: Angel Justiz-Vaillant 2020. ELISA for quantification of IL-18 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj3hkqj6 Copy   

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Authors: Alan Cone
Group: Ju Lab

Proper citation: Alan Cone 2016. 2x Separating Gel Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.fw6bphe Copy   

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Authors: Justin L Brown, Binta Wold, Rachel N Larson
Summary: This scat survey protocol has been developed for use by National Park Service staff of the Santa Monica Mountains National Recreation Area to facilitate the training of citizen science volunteers to collect and analyze coyote scat for the Los Angeles Urban Coyote Project. This protocol provides instructions for the scat project managers to supervise volunteers and train them on how to:Walk a scat transect and collect scatIdentify coyote scat in the fieldDissect scatIdentify major prey itemsIn addition, sections of this protocol detail actions carried out by NPS staff, such as volunteer supervision and quality control, and the protocol for sterilizing and washing scats. We hope this protocol is an effective tool for others who are interested in starting a citizen science scat collection/dissection project in their own research.

Proper citation: Justin L Brown, Binta Wold, Rachel N Larson 2019. LA Urban Coyote Project Volunteer Training Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.933h8qn Copy   

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Authors: Carlos H
Summary: This protocol describes how to make chemically competent Vibrio natriegens cells.The protocol was described and published by Weinstock et al., 2016

Proper citation: Carlos H 2018. Chemically competent V. natriegens cells. protocols.io https://dx.doi.org/10.17504/protocols.io.pumdnu6 Copy   

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Authors: Zhujun Wei
Group: 2020 iGEM NEFU China
Summary: This protocol includes three different methods of digestion.

Proper citation: Zhujun Wei 2020. Restriction Digest. protocols.io https://dx.doi.org/10.17504/protocols.io.bnwvmfe6 Copy   

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Authors: Randy True
Group: FloodLAMP, XPRIZE Rapid Covid Testing
Summary: The Inactivation Solution includes TCEP, EDTA, NaOH, and water. It should be made in a chemical fume hood, as the TCEP is caustic. It is very straightforward to make if starting with liquid-form TCEP. We have not made it with powder-form TCEP, which is about 30% cheaper at low volume retail prices from Sigma. We have yet to do long-term stability studies; however, we think it's fine stored at room temperature for at least a few weeks. If yellow Binding Solution does not go clear upon addition to an inactivated sample or PBS+Inactivation Soln, suspect that the Inactivation Solution may have degraded, and repeat with another aliquot or freshly-made Inactivation Solution.It has also been suggested by Albert Yu of Brandeis has recommended using a 1.4N NaOH concentration for saliva instead of 1.15N NaOH. NaOH should be scale up by 1.4/1.15 and water down for this.

Proper citation: Randy True 2020. FloodLAMP Inactivation Solution Prep v1.0. protocols.io https://dx.doi.org/10.17504/protocols.io.bk5qky5w Copy   

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Authors: Dennis Dienst, Pia Lindberg, João Rodrigues
Group: CyanoWorld, Chemistry Method Development Community
Summary: This is a quick guide for the preparation of (E)-α-bisabolene samples and external standards in dodecane for GC analysis. This protocol has been established in the Lindberg lab at Ångström laboratory (Uppsala University) for direct analysis of dodecane-based ex-situ extracts from cyanobacterial strains producing (E)-α-bisabolene. The protocol includes the preparation of stock solutions for the internal standard β-caryophyllene (BCP) and the external standard. The preparation of a fresh external standard series is suggested for each sample analysis. The calibration curve is required for quantification of (E)-α-bisabolene in the samples of interest. This protocol is used in combination with the following GC protocol:dx.doi.org/10.17504/protocols.io.kj2cuqe

Proper citation: Dennis Dienst, Pia Lindberg, João Rodrigues 2019. (E)-α-bisabolene GC sample preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.mwdc7a6 Copy   

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Authors: Alba Balletbó
Group: iGEM Wageningen 2019

Proper citation: Alba Balletbó 2019. LB Medium (1 Liter). protocols.io https://dx.doi.org/10.17504/protocols.io.7jshkne Copy   

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Authors: Yiheng Hu
Group: High molecular weight DNA extraction from all kingdoms, Team Schwessinger
Summary: Modified from Benjamin Schwessinger: High quality DNA from Fungi for long read sequencing e.g. PacBio. protocols.ioOptimized for DNA extraction from wheat stripe rust spores.Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.Critical steps to obtain high quality DNA:Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.Do NOT incubate samples with KAc for prolonged time periods

Proper citation: Yiheng Hu 2016. High quality DNA extraction from Fungi_small scale. protocols.io https://dx.doi.org/10.17504/protocols.io.exmbfk6 Copy   

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Authors: Alex Rajewski
Summary: This protocol is to fix freshly collected plant tissue for serial sectioning. Although this protocol is similar to ones used for in situ mRNA hybridization, it should not be used for that purpose as it is not nearly careful/gently enough. This protocol has been tested with tomato, tobacco, and date fruits with good results.

Proper citation: Alex Rajewski 2018. Plant Tissue Fixation. protocols.io https://dx.doi.org/10.17504/protocols.io.rw8d7hw Copy   

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Authors: Eric Edsinger, Reuven Pnini, Natsumi Ono, Ryoko Yanagisawa, Kathryn Dever, Jonathan Miller

Proper citation: Eric Edsinger, Reuven Pnini, Natsumi Ono, Ryoko Yanagisawa, Kathryn Dever, Jonathan Miller 2019. Octopus social tolerance experiment. protocols.io https://dx.doi.org/10.17504/protocols.io.w9nfh5e Copy   

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Authors: Invitrogen

Proper citation: Invitrogen 2020. LR Clonase. protocols.io https://dx.doi.org/10.17504/protocols.io.bfm9jk96 Copy   

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