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CRISPR Editing of Immortalized Cell Lines with RNPs using Neon Electroporation Resource Report Resource Website |
Synthego | DOI:10.17504/protocols.io.spredm6 | Synthego | This protocol describes how to deliver ribonucleoprotein (RNP) complexes that consist of purified Cas9 nuclease duplexed with chemically modified synthetic single guide RNA (sgRNA) to immortalized adherent or suspension cells. RNP delivery is accomplished using the Thermo Fisher NeonTM Transfection System. A reference for electroporation settings for a wide variety of cell types is included. A option for knock-in is included. Chemically modified sgRNAs are designed to resist exonucleases and innate intracellular immune cascades that can lead to cell death. Synthego chemically modified synthetic sgRNAs are of exceptional purity and consistently drive high editing frequencies. | Synthego | https://www.synthego.com/resources/immortalized-cell-neon-electroporation-protocol | 1 | 2018 | Synthego 2018. CRISPR Editing of Immortalized Cell Lines with RNPs using Neon Electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.spredm6 | 2021-04-15 09:15:43 | ||
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Unix and Bioinformatics Resource Report Resource Website |
Benjamin Tully and Ken Youens-Clark | DOI:10.17504/protocols.io.fitbken | ECOGEO | This protocol details the use of various unix commands commonly used in bioinformatics. | EARTHCUBE OCEANOGRAPHY AND GEOBIOLOGY ENVIRONMENTAL 'OMICS | 3 | 2016 | Benjamin Tully and Ken Youens-Clark 2016. Unix and Bioinformatics. protocols.io https://dx.doi.org/10.17504/protocols.io.fitbken | 2021-04-15 09:15:15 | |||
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Fluorescence analysis of leaf discs using CF imager Resource Report Resource Website |
Isla Causon, Steven Burgess | DOI:10.17504/protocols.io.rtyd6pw | , University of Illinois at Urbana-Champaign | 1 | 2018 | Isla Causon, Steven Burgess 2018. Fluorescence analysis of leaf discs using CF imager. protocols.io https://dx.doi.org/10.17504/protocols.io.rtyd6pw | 2021-04-15 09:15:15 | |||||
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Protocol for derivatization and determination of structural monosaccharides in crude fungal exopolysaccharide Resource Report Resource Website |
Tayebeh Fooladi, Mohammad Reza Soudi, Majid M. Heravi, Sanaz Karimian | DOI:10.17504/protocols.io.5j8g4rw | .justify:after { content: ""; display:inline-block; width: 100%; } Polysaccharides are, composed of monomeric simple sugars exhibiting substantial diversity for functional groups, found being suitable for vast biological activities in different living organisms. Difficulties in isolation, purification by professional methods and using costly equipment are the multiple challenges of characterizing the nature and properties of these important components of living cells. Several methods have been applied for the qualitative analysis of the carbohydrates. However, GC and GC-MS are the most appropriate techniques ever used to analyze complex structures of polysaccharide. Notably, so far, no commercial kit has been developed and offered, particularly for derivatization of polysaccharides. This study aims to confer a modified protocol for suitable derivatization of structurally unknown polysaccharides. The fungal exopolysaccharide from Neopestalotiopsis sp. has been used as the sample in this study. The study has established the multiple manual steps of the alditol acetate derivatization. The modifications have been highlighted as follows: Trifluoroacetic acid (TFA) has been used instead of conventional trichloroacetic acid for hydrolyzing the crude exopolysaccharide. The standard sugars were treated separately, but not in the complex mixture. The simplicity of the method, using ordinary instruments and short time accomplishment of procedures, collectively makes this technique as the most applicable in a common research laboratory where no more sophisticated equipment is available. | Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Iran, Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Iran, Department of Chemistry, School of Science, Alzahra University, Iran, Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Iran | 1 | 2019 | Tayebeh Fooladi, Mohammad Reza Soudi, Majid M. Heravi, Sanaz Karimian 2019. Protocol for derivatization and determination of structural monosaccharides in crude fungal exopolysaccharide. protocols.io https://dx.doi.org/10.17504/protocols.io.5j8g4rw | 2021-04-15 09:15:42 | ||||
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Release of nucleic acids with heat, chelator, and detergent Resource Report Resource Website |
Grieg F. Steward and Alexander I. Culley | DOI:10.17504/protocols.io.dsc6av | VERVE Net | If one has a purified stock of viruses obtained, for example, by banding in a buoyant density gradient, or even a relatively pure viral concentrate obtained by size fractionation, it is possible to release the DNA in a high molecular weight form suitable for some applications (e.g., pulsed-field gel electrophoresis [PFGE] for sizing or probing, or nucleic acid quantification by fluorescence) relatively simply. This method involves exchanging the buffer in which the viruses are suspended with one containing EDTA and SDS, followed by heating. The method is similar to that described previously (Steward 2001), but with the optional addition of detergent to facilitate disintegration of the viral capsid.This is a protocol from: Steward, G. F. and A. I. Culley. 2010. Chapter 16: Extraction and purification of nucleic acids from viruses. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7Please see the published manuscript for additional information. | Manual of Aquatic Viral Ecology | http://www.aslo.org/books/mave/MAVE_154.pdf | 1 | 2015 | Grieg F. Steward and Alexander I. Culley 2015. Release of nucleic acids with heat, chelator, and detergent. protocols.io https://dx.doi.org/10.17504/protocols.io.dsc6av | 2021-04-15 09:15:43 | ||
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Immunohistochemistry Protocol for Frozen Sections Resource Report Resource Website |
Kelsey Knight | DOI:10.17504/protocols.io.tkfektn | BioLegend | The following is a general procedure guide for preparation and staining of acetone-fixed frozen tissues using a purified, unconjugated primary antibody, biotinylated secondary antibody and streptavidin-horseradish peroxidase (Sav-HRP) and DAB detection system. Because each antigen differs in terms of requirement for fixation, amplification step, etc., it is not possible to write an inclusive protocol that will work for all antigens. The user must determine optimal conditions for each antigen of interest. Many protocols for staining individual antigens, as well as useful tips and troubleshooting guides for immunohistochemistry, can be found at the IHC World web site (http://www.ihcworld.com/). | BioLegend | https://www.biolegend.com/protocols/immunohistochemistry-protocol-for-frozen-sections/4288/ | 2 | 2018 | Kelsey Knight 2018. Immunohistochemistry Protocol for Frozen Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.tkfektn | 2021-04-15 09:15:16 | ||
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ExFISH - Cultured Cells Resource Report Resource Website |
Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden | DOI:10.17504/protocols.io.n32dgqe | Human Cell Atlas Method Development Community | The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine. | Massachusetts Institute of Technology, Massachusetts Institute of Technology, University of Pennsylvania, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University, University of Pennsylvania, Massachusetts Institute of Technology | http://expansionmicroscopy.org/ExFISHV1.0.docx | 1 | 2018 | Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden 2018. ExFISH - Cultured Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n32dgqe | 2021-04-15 09:15:43 | ||
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Anti-c-Myc Tag (9E10) Affinity Gel Protocol Resource Report Resource Website |
Sam Li | DOI:10.17504/protocols.io.97ih9ke | BioLegend | Catalog Number: 658502Storage Temperature: 2°C-8°CProduct Description: Anti-c-Myc Tag (9E10) Affinity Gel consists of anti-c-myc monoclonal antibody (clone 9E10), covalently immobilized onto 6% high density glyoxal agarose beads. The affinity resin can be used in affinity purifying and immunoprecipitation of c-Myc-tagged fusion protein.Binding Specificity: Mouse monoclonal antibody 9E10 recognizes the c-myc epitope N-EQKLSEEDL-C and is purified through Protein G chromatography. The antibody conjugated affinity resin can bind to epitope at N-terminal, C-terminal, and internal locations of a fusion protein. The binding capacity is greater than 0.5mg per ml of c-myc agarose resin.Reagent: Anti-c-Myc Agarose Affinity Gel is supplied as a 50% suspension in phosphate-buffered saline, pH 7.2, containing 0.09% sodium azide.Precautions and Disclaimer: This product is for research use only. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.Storage/Stability: Anti-c-Myc Agarose Affinity Gel should be stored between 2°C and 8°C for maximum stability. The unopened product is stable for one year when stored as indicated. After use, the resin should be regenerated and stored in TBS or PBS buffer (pH 7.2) containing 0.09% sodium azide. | BioLegend | https://www.biolegend.com/protocols/anti-c-myc-tag-9e10-affinity-gel-protocol/4297/ | 2 | 2019 | Sam Li 2019. Anti-c-Myc Tag (9E10) Affinity Gel Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.97ih9ke | 2021-04-15 09:15:15 | ||
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Changes in bioelement contents of summer and winter western honeybees (Apis mellifera) induced by Nosema ceranae infection Resource Report Resource Website |
Aneta Ptaszynska | DOI:10.17504/protocols.io.qwedxbe | Ptaszyńska AA, Gancarz M, Hurd PJ, Borsuk G, Wiącek D, Nawrocka A, Strachecka A, Załuski D, Paleolog J, Changes in the bioelement content of summer and winter western honeybees () induced by infection. PLoS ONE 13(7). doi: 10.1371/journal.pone.0200410 | Department of Botany and Mycology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, 19 Akademicka st., 20-033 Lublin, Poland | https://doi.org/10.1371/journal.pone.0200410 | 1 | 2018 | Aneta Ptaszynska 2018. Changes in bioelement contents of summer and winter western honeybees (Apis mellifera) induced by Nosema ceranae infection. protocols.io https://dx.doi.org/10.17504/protocols.io.qwedxbe | 2021-04-15 09:15:16 | |||
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Optimization of blood pressure management after acute ischemic stroke and its prognostic significance: Prospective, randomized, open, blinded outcome evaluation, and feasibility Trial Resource Report Resource Website |
Beom Joon Kim | DOI:10.17504/protocols.io.9nqh5dw | Optimization of blood pressure management after acute ischemic stroke and its prognostic significance: Prospective, randomized, open, blinded outcome evaluation, and feasibility Trial [BOSS trial]; NCT03024476A phase II clinical trial protocol written in Korean language. Apologize not providing a translation. | Kim BJ, Park J, Park TH, Kim J, Lee J, Lee K, Lee J, Chae JE, Thabane L, Lee J, Bae H (2020) Remote blood pressure monitoring and behavioral intensification for stroke: A randomized controlled feasibility trial. PLoS ONE 15(3): e0229483. doi: 10.1371/journal.pone.0229483 | Seoul National University Bundang Hospital | https://clinicaltrials.gov/ct2/show/NCT03024476 | 1 | 2019 | Beom Joon Kim 2019. Optimization of blood pressure management after acute ischemic stroke and its prognostic significance: Prospective, randomized, open, blinded outcome evaluation, and feasibility Trial. protocols.io https://dx.doi.org/10.17504/protocols.io.9nqh5dw | 2021-04-15 09:15:16 | ||
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Quantifying how FAIR is Hong Kong: The Hong Kong Shareability of Hong Kong University Research Experiment Resource Report Resource Website |
Scott Edmunds, Jesse Xiao, Hongling Zhou | DOI:10.17504/protocols.io.6x7hfrn | BGI | Using 59 HKU students undertaking the data curation module of the Master of Science in Library and Information Management (MLIM) course taught by some of the GigaScience team, a crowdsourced literature curation exercise was carried out to assess the data availability of HKU's papers published in PLOS One and Scientific Reports journals between 2016-2018. This experiment can be continued at other institutions, and has been presented to allow a teacher of a similar course to set this up for their own students.This collection includes protocols for teachers trying to set up this exercise at a different university, and the protocol for the individual students carrying it out. Alongside a spreadsheet to input the results. | GigaScience/BGI Hong Kong/Bauhinia Genome, GigaScience/BGI Hong Kong, GigaScience/BGI | 1 | 2019 | Scott Edmunds, Jesse Xiao, Hongling Zhou 2019. Quantifying how FAIR is Hong Kong: The Hong Kong Shareability of Hong Kong University Research Experiment. protocols.io https://dx.doi.org/10.17504/protocols.io.6x7hfrn | 2021-04-15 09:15:42 | |||
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Make potassium orthophosphate solution Resource Report Resource Website |
Gurdon Institute media kitchen | DOI:10.17504/protocols.io.5yeg7te | Wellcome Trust / Cancer Research UK Gurdon Institute | 7 | 2019 | Gurdon Institute media kitchen 2019. Make potassium orthophosphate solution. protocols.io https://dx.doi.org/10.17504/protocols.io.5yeg7te | 2021-04-15 09:15:15 | |||||
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Lithium chloride purification, a rapid and efficient technique to purify RNA from DSS-treated tissues Resource Report Resource Website |
Emilie Viennois | DOI:10.17504/protocols.io.kk3cuyn | Dextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis. However, the mechanisms of action of DSS remain poorly understood, and observations by our laboratory and other groups indicate that DSS contamination of colonic tissues from DSStreated mice potently inhibits the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplification of mRNA. A prior study used poly-A-mediated mRNA purification to remove DSS from RNA extracts, but we herein report a second efficient and cost-effective approach to counteract this inhibition, using lithium chloride precipitation to entirely remove DSS from RNAs. We also explored how DSS interferes with qRT-PCR process, and we report for the first time that DSS can alter the binding of reverse transcriptase to previously primed RNA and specifically inhibits the enzymatic activities of reverse transcriptase and Taq polymerase in vitro. This likely explains why DSS-treated colonic RNA is not suitable to qRT-PCR amplification without a previous purification step. In summary, we provide a simple method to remove DSS from colonic RNAs, and we demonstrate for the first time that DSS can inhibit the activities of both polymerase and reverse transcriptase. In order to reliably analyze gene expression in the colonic mucosa of DSS-treated mice, the efficiency rate of qRT-PCR must be the same between all the different experimental groups, including the water-treated control group, suggesting that whatever the duration and the percentage of the DSS treatment, RNAs must be purified. | Gerorgia State University | 2 | 2017 | Emilie Viennois 2017. Lithium chloride purification, a rapid and efficient technique to purify RNA from DSS-treated tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.kk3cuyn | 2021-04-15 09:15:15 | ||||
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modelling stiffness of lower limb by predictive classifier to control stiffness of prosthec leg Resource Report Resource Website |
Mohd Rizwan Jafar | DOI:10.17504/protocols.io.memc3c6 | this experiment describes a process to deveop a classifier which can be used to modulate the stiffness of prosthetic leg based on the movement of hands, elbows and position of the other leg of the amputee | Delhi College of Engineering (DCE) | 1 | 2017 | Mohd Rizwan Jafar 2017. modelling stiffness of lower limb by predictive classifier to control stiffness of prosthec leg. protocols.io https://dx.doi.org/10.17504/protocols.io.memc3c6 | 2021-04-15 09:15:42 | ||||
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Setting up Experimental Cultures for Large-scale One-step Phage Infection of Cyanobacteria Resource Report Resource Website |
Sarah Giuliani | DOI:10.17504/protocols.io.fhqbj5w | Coleman Lab | Experiment purpose is to monitor the time-course of a large-scale infection of host cyanobacteria by phage under variable media conditions and obtain samples for proteomic and transcriptomic analysis.As a minimum, prepare 12 sample bottles for cells infected with phage and control phage lysate, under 2 different media treatments, with triplcates of each unique Phage/Treatment combination. | Coleman Lab - University of Chicago, Department of the Geophysical Sciences | 1 | 2016 | Sarah Giuliani 2016. Setting up Experimental Cultures for Large-scale One-step Phage Infection of Cyanobacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.fhqbj5w | 2021-04-15 09:15:15 | |||
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NLP screening (dot blot) Resource Report Resource Website |
Andreea S | DOI:10.17504/protocols.io.bkbfksjn | iGEM Groningen 2020 | In order to determine if the His-tagged NLP is expressed and secreted by the bacteria, we plan to use the dot blot procedure with anti-His tag anti-bodies. Dot blot is an immunological technique used for detecting proteins directly from the culture supernatant (without gel separation). Thus, the samples are directly spotted on the membrane, making it a high throughput procedure ideal for testing different secretion signals. | University of Groningen | 1 | 2020 | Andreea S 2020. NLP screening (dot blot). protocols.io https://dx.doi.org/10.17504/protocols.io.bkbfksjn | 2021-04-15 09:15:15 | |||
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RNA extration for the R. crenulata genome Resource Report Resource Website |
Yuanyuan Fu, Liangwei Li, Shijie Hao, Rui Guan, Guangyi Fan, Chengcheng Shi, Haibo Wan, Wenbin Chen, He Zhang, Guocheng Liu, Jihua Wang, Lulin Ma, Jianling You, Xuemei Ni, Zhen Yue, Xun Xu, Xiao Sun, Xin Liu, Simon Ming-Yuen Lee | DOI:10.17504/protocols.io.hrjb54n | GigaScience Press | This protocol is used to clarify the process of RNA extration for our R. crenulata genome. | Fu Y, Li L, Hao S, Guan R, Fan G, Shi C, Wan H, Chen W, Zhang H, Liu G, Wang J, Ma L, You J, Ni X, Yue Z, Xu X, Sun X, Liu X, Lee SM, Draft genome sequence of the Tibetan medicinal herb . GigaScience 6(6). doi: 10.1093/gigascience/gix033 | Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China, Institute of Chinese Medical Sciences, University of Macau, China | https://doi.org/10.1093/gigascience/gix033 | 1 | 2017 | Yuanyuan Fu, Liangwei Li, Shijie Hao, Rui Guan, Guangyi Fan, Chengcheng Shi, Haibo Wan, Wenbin Chen, He Zhang, Guocheng Liu, Jihua Wang, Lulin Ma, Jianling You, Xuemei Ni, Zhen Yue, Xun Xu, Xiao Sun, Xin Liu, Simon Ming-Yuen Lee 2017. RNA extration for the R. crenulata genome. protocols.io https://dx.doi.org/10.17504/protocols.io.hrjb54n | 2021-04-15 09:15:42 | |
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Th2 Polarization of Mouse CD4+ Cells Resource Report Resource Website |
Kelsey Knight | DOI:10.17504/protocols.io.tnkemcw | BioLegend | BioLegend | https://www.biolegend.com/protocols/th2-polarization-of-mouse-cd4--cells-protocol/4238/ | 2 | 2018 | Kelsey Knight 2018. Th2 Polarization of Mouse CD4+ Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.tnkemcw | 2021-04-15 09:15:15 | |||
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UC Davis - Luminex/Multiplex Resource Report Resource Website |
Lori Haapanen | DOI:10.17504/protocols.io.56wg9fe | Mouse Metabolic Phenotyping Centers | This core will provide services for quantification and comparison in animal models for hormone measurements; kidney function analytes; lipid metabolism analytes; liver function analytes; miscellaneous protein analytes; and immune function including cytokines/chemokines. The Luminex assay enables the quantification of all or any combination of analytes for which there are assay beads in tissue/cell lysate, tissue culture supernatant samples and plasma/serum. For example, cytokines and chemokines are peptides, which act as regulators in normal and pathological conditions, effecting interactions between cells as well as regulating processes occurring in the extracellular environment. | University of California, Davis | https://mmpc.org/shared/document.aspx?id=116&docType=Protocol | 2 | 2019 | Lori Haapanen 2019. UC Davis - Luminex/Multiplex. protocols.io https://dx.doi.org/10.17504/protocols.io.56wg9fe | 2021-04-15 09:15:15 | ||
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The prognostic role of serum uric acid levels in preeclampsia: a meta-analysis Resource Report Resource Website |
Ioannis Bellos, Vasilios Pergialiotis, Dimitrios Loutradis, Georgios Daskalakis | DOI:10.17504/protocols.io.bcndiva6 | The present meta-analysis will assess the possible association of serum uric acid with the development of preeclampsia. Preeclampsia represents a serious pregnancy complication with increased fetal and maternal morbidity and mortality rates. Its incidence is estimated at approximately 5-8% of pregnancies, although presenting wide variance worldwide. Its pathophysiology is complex; however, it is hypothesized that the combination of poor trophoblast invasion with the release of angiogenic and pro-oxidant mediators into maternal circulation leads to generalized endothelial dysfunction. Prediction of the disease is necessary in order to provide measures of prevention, especially the administration of aspirin; nonetheless, the optimal screening model remains under investigation. All the available literature evidence regarding the prognostic utility of serum uric acid in preeclampsia is planned to be evaluated, by applying no date or language restrictions during the literature search. Result will be pooled by a quantitative meta-analytic approach, planning to subgroup outcomes on the basis of pregnancy trimester, disease severity and onset. The credibility of evidence will be evaluated under the GRADE framework. | National and Kapodistrian University of Athens, National and Kapodistrian University of Athens, National and Kapodistrian University of Athens, National and Kapodistrian University of Athens | 1 | 2020 | Ioannis Bellos, Vasilios Pergialiotis, Dimitrios Loutradis, Georgios Daskalakis 2020. The prognostic role of serum uric acid levels in preeclampsia: a meta-analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bcndiva6 | 2021-04-15 09:15:42 |
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