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Authors: Lawrence A. Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Numerous unpublished studies indicate that Euplotes crassus is resistant to extremely high concentrations of commonly used selective agents when grow in artificial seawater. We have found that when E. crassus is grown in 1 part artificial seawater + 9 parts 0.3 M glucose, they are much more sensitive to a number of selective agents. For example, 1080 ug/ml paromomycin in artificial seawater only inhibited growth, as little as 120 ug/ml was effective in killing cells when grown with 0.3 M glucose. Similarly, G418 at 400 ug/ml had little effect in seawater, but as little as 100 ug/ml blocked growth in 0.3 M glucose. This may not prove true for all selective agents, as we saw no effect with paclitaxel up to 100 uM under either growth condition.
Proper citation: Lawrence A. Klobutcher 2017. Increased sensitivity of Euplotes crassus to selective agents using 0.3 M glucose-based culture conditions.. protocols.io dx.doi.org/10.17504/protocols.io.gwnbxde Copy
Authors: Alexandra Ehl, David Frommholz, Nadine Stefanczyk
Summary: Purification Guide for the Isolation of Antibodies with ChroPlate Filtration Plates by DALEX Biotech.Easy and quick high throughput antibody purification from various sources and species.Each well of the ChroPlate has a binding capacity of > 1 mg (tested with human polyclonal Ig, binding varies between species and clones).The proprietary resin does not shrink or swell in aqueous buffers.High pressure stability.pH stability short term 2 - 8, long term 3 - 8.Excellent thermal stability up to 15 minutes at 80 °C in aqueous buffers at neutral pH.Can be dried for long term storage (80 °C for > 2 h).
Proper citation: Alexandra Ehl, David Frommholz, Nadine Stefanczyk 2018. ChroPlate - ProteinG. protocols.io dx.doi.org/10.17504/protocols.io.uyjexun Copy
Authors: Mark DeWitt
Group: CornLab
Summary: This protocol describes how to extract and amplify DNA from genome-edited cells, and sequence the resulting amplicons using Illumina MiSeq NGS.
Proper citation: Mark DeWitt 2017. Preparation of PCR amplicons from edited cells for deep sequencing. protocols.io dx.doi.org/10.17504/protocols.io.hdsb26e Copy
Authors: 宏亮 董
Group: 2019 iGEM NEFU_China
Summary: This PCR method can be used to screen for inserted target genes or DNA sequencing analysis.
Proper citation: 宏亮 董 2019. Colony PCR. protocols.io dx.doi.org/10.17504/protocols.io.7h2hj8e Copy
Authors: Erin Garza, Vincent Bielinski
Group: Protist Research to Optimize Tools in Genetics (PROT-G), JCVI West Protocols
Summary: A high-throughput method for measuring β-glucuronidase (GUS) activity in the diatom Phaeodactylum tricornutum. This protocol has been optimized for 250 μl volumes. For larger volumes see the following protocol dx.doi.org/10.17504/protocols.io.hefb3bn, which this protocol was based off of.
Proper citation: Erin Garza, Vincent Bielinski 2020. High-Throughput Beta-glucuronidase (GUS) assay for Phaeodactylum tricornutum. protocols.io dx.doi.org/10.17504/protocols.io.bbexijfn Copy
Authors: Integrated Islet Distribution Program
Group: Integrated Islet Distribution Program
Summary: This Standard Operating Procedure is adapted from the work of the 'National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities'' following the SOP cited in the document 'Purified Human Pancreatic Islet - Viability Estimation of Islet Using Fluorescent Dyes (FDA/PI): Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium'This SOP defines the procedure for assessment of viability of human isolated islet preparations, which include endocrine and exocrine tissue, for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP). This protocol is written to assist the participating islet isolation centers and investigators who are part of this program.Fluorescein Diacetate/ Propidium Iodide (FDA)/(PI) Viability Assay is a rapid fluorometric method to test the integrity of the plasma membrane simultaneously using inclusion and exclusion dyes; the assay differentiates between viable and nonviable cells and is, consequently, used for determination of viability of islet preparations.
Proper citation: Integrated Islet Distribution Program 2019. Viability Estimation of Islets for Distribution Using Inclusion and Exclusion Fluorescent Dyes (FDA/PI). protocols.io dx.doi.org/10.17504/protocols.io.43kgykw Copy
Authors: lisa.diamond
Group: XPRIZE Rapid Covid Testing
Summary: Pinpoint's nanosensor-based platform detects SARS-CoV-2 antigen by (1) measuring baseline current through our functionalized nanosensor chip in ordinary PBS buffer; (2) measuring the change in current at specific voltage ranges after insertion of the specimen as a result of binding between probe aptamer and target antigen; (3) computing delta between these two measurements. These measurements and computations are performed internally in quadruplicate, with four separately measured nanosensor channels. Consolidated results are reported to the user as {Detected; Not Detected; Inconclusive; or Error}.
Proper citation: lisa.diamond 2020. Pinpoint Science 30-Second Covid-19 Test Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bkxmkxk6 Copy
Authors: Oliver Fiehn
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Description of standard setting for analysis of derivatized samples ready for injection into a Leco Pegasus III or Pegasus IV gas chromatograph-time of flight mass spectrometer.References: Fiehn O, Wohlgemuth G, Scholz M, Kind T, Lee DY, Lu Y, Moon S, Nikolau BJ (2008) Quality control for plant metabolomics: Reporting MSI-compliant studies. Plant J. 53, 691-704
Proper citation: Oliver Fiehn 2019. UC Davis - Metabolomics: Primary metabolites by GC-TOF MS. protocols.io dx.doi.org/10.17504/protocols.io.64nhgve Copy
Authors: Lyle J. Arnold Ph.D., Stella M. Sung Ph.D.
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: This protocol is for the Aegea Biotechnologies rapid PCR-based SARS-CoV-2 test. This assay uses patented "Switch-Blocker" technology as well as taqman probes to test for presence of SARS-CoV-2 and simultaneously orthogonally validate. A single amplification reaction is performed, and "Switch Blocker" is used on the forward strand and taqman is used on the reverse strand. The assay design has high sensitivity & specificity--single nucleotide level. Moreover, it is able to detect the SARS-CoV-2 L strain vs the SARS-CoV-2 S strain. The test can be adapted to point of care (Roche LIAT) as well as for different SARS-CoV-2 strains as the virus mutates. A next generation version of the assay could identify the presence of the SARS-CoV-2 L/S strains vs. influenza A/B. Finally, because of the sensitivity and specificity, the Aegea PCR-based SARS-CoV-2 test should be able to use saliva samples, and it is suitable for pooled testing. This protocol is designed for high throughput PCR (96 or 384 well plate formats).Keywords: PCR, COVID-19, coronavirus, SARS-CoV-2, high throughput, multiplex, Switch-Blocker, taqman, high sensitivity, high specificity, accurate, pooling, saliva, strain types, L-strain, S-strain, combination SARS-CoV-2 and influenza
Proper citation: Lyle J. Arnold Ph.D., Stella M. Sung Ph.D. 2020. Aegea Biotechnologies rapid PCR SARS-CoV-2 test (high sensitivity & specificity; able to detect different strain types). protocols.io dx.doi.org/10.17504/protocols.io.bk2dkya6 Copy
Authors: Jorge Frias, Nelson Simões
Summary: Potato-based medium for culture of Drosophila melanogaster.
Proper citation: Jorge Frias, Nelson Simões 2020. Fruit fly potato culture media. protocols.io dx.doi.org/10.17504/protocols.io.bei3jcgn Copy
Authors: Laszlo Irinyi, Lana Pasic, Wieland Meyer
Proper citation: Laszlo Irinyi, Lana Pasic, Wieland Meyer 2019. DNA extraction from sputum. protocols.io dx.doi.org/10.17504/protocols.io.6enhbde Copy
Authors: Jeff Hodgin
Group: Mouse Metabolic Phenotyping Centers
Summary: Picrosirius red staining (also called Sirius red staining) highlights fibrosis by staining collagen (collagen 1 and III fibers) fibers in paraffin-embedded kidney tissue sections. Sirius red stained collagen appears red by light microscopy. When polarized, the birefringence is highly specific for collagen. The larger collagen fibers are bright yellow or orange, and the thinner ones, including reticular fibers, are green.
Proper citation: Jeff Hodgin 2019. U Michigan - Sirius Red staining. protocols.io dx.doi.org/10.17504/protocols.io.56tg9en Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes plasmid modification by annealed oligo cloning. To see the full abstract and additional resources, please visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Plasmid Modification by Annealed Oligo Cloning. protocols.io dx.doi.org/10.17504/protocols.io.bawkifcw Copy
Authors: Berthold Bivigou-Mboumba, Sandrine François-Souquière, Luc Deleplancque, Jeanne Sica, Augustin Mouinga-Ondémé, Marie Amougou-Atsama, Marie Laure Chaix, Richard Njouom, François Rouet
Summary: HBV DNA Real Time Quantification according to ARNS 12187 projectUsing the Arrow extractor (NorDiag, Biotrin International, Ireland), DNA was extracted from 240 μl of plasma, pre-treated with10 μl of proteinase K, using the Arrow Viral NA extraction kit, according to the manufacturer’s instructions during 45 min. Template DNA was eluted into 60 μl of kit elution buffer. Besides clinical samples, quantification standard (Acrometrix HBV Panel, Acrometrix, Menica, CA, USA) was also extracted for each run with the same protocol and1:10 diluted (from 50,000,000IU/mL to50IU/mL). For amplification, we used a primers/probe set designed under the auspices of “Agence Nationale de Recherches sur le SIDA et les hépatites virales” (ANRS 12187 project) and targeting a conserved region in the HBVS gene (nucleotide (nt) positions, 379–426). All runs were performed in a 50-μl volume containing DNA extract (10 μl), Master Mix (Platinum UGD, USA) (25 μl), pure water (HyClone Pure Water, Thermo Fisher Scientific, Waltham, MAUSA)(12.5 μl), forward primHBV1 (5’-GTGTCTGCGGCGTT TTATCA-3’) and reverse primHBV2 (5’-AGGCATAGCAGCAGGAT GAA-3’) primers at 10 μM (1μl each) and probe (5’FAM-TGCGGCGTTTTATCAT-MGB3’) at 5μM (0.5 μl). Each reaction consisted of: 2min at50°C and 10min at95°C; followed by 50 cycles of 15sec at 95°C and 1 min at 60°C each. The lower limit of quantification (LLOQ) of our technique was 100IU/mL and the lower limit of detection (LLOD) was 50IU/mL.
Proper citation: Berthold Bivigou-Mboumba, Sandrine François-Souquière, Luc Deleplancque, Jeanne Sica, Augustin Mouinga-Ondémé, Marie Amougou-Atsama, Marie Laure Chaix, Richard Njouom, François Rouet 2018. HBV DNA Real Time Quantification according to ARNS 12187 project. protocols.io dx.doi.org/10.17504/protocols.io.kw4cxgw Copy
Authors: Nick Tang
Group: VERVE Net, Upton-Lab
Summary: Viral Bioinformatic Resource CentreProvide databases of viral genomic information. Please check the Organisms menu to see which viruses we support: we’re now focusing on large DNA viruses
The VOCs (Virus Orthologous Clusters) database is at the heart of our system.
The database links directly to integrated tools for comparative analyses.
VOCs sorts genes into ortholog clusters (e.g. RNA polymerase) to simplify data retrieval.
Provide easy access to the genes, gene families, and genomes of the different virus families. via a unique series of powerful Java tools that support multiple computer platforms (see VBRC Tools menu).
design and build software to tackle specific bioinformatics/virology problems, often in collaboration with virologists.
Rally the research community to provide expert curation of these viral genomes by: Adding value to GenBank sequences through enhancing and updating genome annotations
Linking to research reviews/papers for the research community.
Collaborate with researchers to help on specific bioinformatics problems, e.g. Custom searches of the databases
Building new features into our tools
Help with genome annotation
Proper citation: Nick Tang 2016. VBRC Base By Base: Switching between Mouse Modes. protocols.io dx.doi.org/10.17504/protocols.io.ecpbavn Copy
Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: Voluntary wheel running provides detailed information on the running capacity and activity patterns of control, treated or mutant mice. Running activity is evaluated from the number of rotations of a stationary wheel detected by sensors attached to running wheel. Running wheel activity provides information regarding when the animal runs, for how long and far, providing temporal and longitudinal data. This system may also be used as a means of providing voluntary exercise in metabolic or exercise based studies.
Proper citation: Jennifer Rutkowsky 2019. UC Davis - Running Wheel. protocols.io dx.doi.org/10.17504/protocols.io.yv9fw96 Copy
Authors: John H. Paul and Markus Weinbauer
Group: VERVE Net
Summary: This protocol gives a method for isolating temperate phages from marine viral concentrations. This protocol uses the conventional plaque agar overlay and looking for turbid or haloed plaques, a hallmark of temperate phages.Paul, J. H., and M. Weinbauer. 2010. Detection of lysogeny in marine environments, p. 30–33. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.Please see the full chapter for additional details.
Proper citation: John H. Paul and Markus Weinbauer 2016. Isolation of temperate phages by plaque agar overlay. protocols.io dx.doi.org/10.17504/protocols.io.dpd5i5 Copy
Authors: Carter Palmer, Christine Liu, Jerold Chun
Group: BICCN
Summary: This protocol is intended to be used for the isolation of nuclei from fresh-frozen brain tissue in preparation for analysis by Single-Nucleus Chromatin Accessibility and mRNA Expression sequencing (SNARE-seq). It has been applied to tissues from mouse, marmoset, and human.
Proper citation: Carter Palmer, Christine Liu, Jerold Chun 2020. Nuclei Isolation for SNARE-seq2 . protocols.io dx.doi.org/10.17504/protocols.io.8tvhwn6 Copy
Authors: G-Biosciences
Group: G-Biosciences
Proper citation: G-Biosciences 2016. Collection of FOCUS™ Mitochondria Kit protocols. protocols.io dx.doi.org/10.17504/protocols.io.e8tbhwn Copy
Authors: Alan Cone
Group: Ju Lab
Proper citation: Alan Cone 2016. Protein Coomassie Blue Staining. protocols.io dx.doi.org/10.17504/protocols.io.fw8bphw Copy
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