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Authors: Lauren Segers, Kendall Morris, Sarah Nuding
Group: SPARC

Proper citation: Lauren Segers, Kendall Morris, Sarah Nuding 2019. Morris USF Lab protocol DEMO. protocols.io https://dx.doi.org/10.17504/protocols.io.5gbg3sn Copy   

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Authors: Tomoko Matsuda
Summary: This protocol is for RNA Isolation from suspended Chinese hamster ovary (CHO) cells. Wash cells once in 1× PBS and isolate RNA using Qiashredder columns and the RNeasy mini kit. In the case of RNA-Seq, which reveals the presence and quantity of RNA in a biological sample at any moment, it is necessary to consider that gene expression responds over a short time interval (several seconds to a few minutes) in many organisms. Therefore, to isolate RNA that accurately reflects the transcriptome at the point of harvest, raw biological samples should be processed by lysing and homogenizing in RNA lysis buffer containing guanidine thiocyanate (buffer RLT, RNA lysis buffer, QIAGEN) as soon as possible (within 15 minutes, if possible).

Proper citation: Tomoko Matsuda 2021. RNA isolation from suspended animal cells, cDNA library construction, and RNA-Seq for gene expression analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.57ng9me Copy   

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Authors: Evripidis Gkanias, Benjamin Risse, Michael Mangan, Barbara Webb
Summary: InsectVisionComputer Vision Toolbox for Insect VisionVersion 1.0.1This version has been created as support for the article:Gkanias E., Risse B., Mangan M., and Webb B. (2019) From skylight input to behavioural output: a computational model of the insect polarised light compass. PLOS Computational Biology.To be able to run all the experiments and replicate the results presented in the article, the compmodels package is needed. If you don't have access to this package, please contact the authors.Clone both repositories and set the compmodels as a dependence to the insectvision package.To see the results, it is not necessary to run the code. By simply opening the notebooks/plos.ipynb file, the plots should be automatically generated for you to observe them.To create the plots by yourself, you need to start a Jupyter notebook kernel at the root of the package. Then run the notebooks/plos.ipynb file, which already contains all the plots and the respective code to replicate them. Some plots (especially the ones related to the global optimisation) may need a long time to run; this does not mean that they do not work.All the code has been implemented by Evripidis Gkanias.You can go the github repository by following this link. The release version is attached and it can also be found here.Copyright © 2019 Gkanias el al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Proper citation: Evripidis Gkanias, Benjamin Risse, Michael Mangan, Barbara Webb 2019. Insect Vision 1.0.1. protocols.io https://dx.doi.org/10.17504/protocols.io.y9hfz36 Copy   

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Authors: Jeremy Kamil
Summary: I learned this Colloidal Coomassie Blue staining recipe and protocol when I was a postdoc at Harvard Medical School.. Rumor has it, a postdoc reverse engineered a “safe stain” from a leading commercial supplier. It’s a really great recipe: much more sensitive, faster, and easier to use than traditional methanol based coomassie staining protocols you see in Cold Spring Harbor books, like by Maniatis etc.. Saves a ton of money compared to buying this stuff from commercial vendors. It is downstream compatible with mass spec and most other things you'd want to do with gel slices.

Proper citation: Jeremy Kamil 2020. Colloidal Coomassie Blue Stain: Recipe and Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bmmtk46n Copy   

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Authors: Abebe A. Fola , Jack Dorman, Maria Levy, Ilinca Ciubotariu, Giovanna Carpi
Summary: Genomic DNA (gDNA) extracts from human dried blood spots (DBS) are commonly used for infectious diseases surveillance, genomic epidemiology studies of targeted pathogens, and Biobanking. In particular, DBS samples are routinely collected during cross-sectional and large-scale epidemiological studies (i.e. Malaria Indicator Survey) in malaria-endemic countries for malaria surveillance and genomic studies. DBS samples have multiple advantages over venous blood, including less invasive sample blood collection (finger prick) and the recommended short- and long-term storage at room temperature, which facilitates sample transportation (domestically or internationally). Here we present an optimized high throughput gDNA extraction protocol to recover Plasmodium gDNA from human DBS (collected on Whatman filter paper) using field samples from Zambia and stored at room temperature with desiccant pellet (up to 2 years). Using the QIAcube HT instrument and QIAmp DNA 96-well kit, this protocol allows for gDNA extraction from human DBS samples in a manner that is automated, reproducible, and fast when compared to other methods. After gDNA extraction is completed, genomic DNA quantity and integrity is assessed using Qubit 1x dsDNA High Sensitivity Assay and Genomic DNA ScreenTape on the Agilent TapeStation 4150, respectively. In this case, gDNA samples that were obtained from field collected human DBS samples and extracted using this optimized protocol were then used for Plasmodium falciparum whole-genome sequencing by hybrid capture.Using this protocol, we first compared gDNA concentration between 2X6mm and 3X3mm Uni Core full punch (100% blood punch) and found a statistically significant difference in DNA concentration obtained from 2x6mm (ng/µL) vs 3x3mm DBS punches (range =0.44-2.01ng/µL) (P-value= 0.0003) with a median increased of ~4-fold in the gDNA yield when using 2x6mm hole punches. See the attached Comparison of DNA concentration 2X6mm vs 3X3mm Uni Core Punch documents. Furthermore, we tested and demostrated that using this protocol with 2X6mm punches, it is feasible to successfully extract sufficient gDNA for P. falciparum whole genome capture and deep sequencing from DBS samples (n=93) that had been stored at RT for up to 2 years with variable level of parasitemia. While generally concentrations were outside the recommended range for the Genomic DNA ScreenTape for the TapeStation DIN calculation, from those samples DIN was estimated, values ranged from 6.2 - 8.7 with average fragment peaks of 35,325 bp, indicating that DNA integrity is suitable for downstream analysis. Becuase these were field DBS samples, we reported high variation in gDNA concentration (ranges= 0.50-4.88ng/µL) which reflected the variation in blood volume and adsorbance across DBS samples. See the attached gDNA concentration for 93 DBS field samples documents.

Proper citation: Abebe A. Fola , Jack Dorman, Maria Levy, Ilinca Ciubotariu, Giovanna Carpi 2020. Optimized HT gDNA extraction from Dried Blood Spot using QIAcube HT for Malaria Genomic Epidemiology Studies. protocols.io https://dx.doi.org/10.17504/protocols.io.bh69j9h6 Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Peroxidase conjugated to proteins L, A and G (SpLAG-HRP) is a new development in the field of biochemistry. This versatile protein binds to more 500 species of animal IgGs. It makes it suitable for the study of zoonosis and the assessment of the antibody production to infectious microorganisms. This immunomarker is not available in a recombinant form as their counterpart SpLA-HRP, SpLG-HRP and SpAG-HRP. It can be used in ELISA, immunohistochemistry, Western blot analysis, Dot blot, and RIA [1,2]. References1. Justiz-Vaillant AA, Akpaka PE, McFarlane-Anderson N, Smikle MF. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.West Indian Med J. 2013;62(1):12-20.2. Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175.

Proper citation: Angel Justiz-Vaillant 2020. Protocol of preparation of a protein-LAG conjugated to horseradish peroxidase by the periodate method.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjk7kkzn Copy   

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Authors: Bonnie Hurwitz, Ken Youens-Clark
Group: VERVE Net, Hurwitz Lab
Summary: Hurwitz, Bonnie L., and Matthew B. Sullivan. "The Pacific Ocean Virome (POV): a marine viral metagenomic dataset and associated protein clusters for quantitative viral ecology." PLoS One 8.2 (2013): e57355.

Proper citation: Bonnie Hurwitz, Ken Youens-Clark 2016. Running PCPipe (protein clustering) in iPlant. protocols.io https://dx.doi.org/10.17504/protocols.io.ef8bbrw Copy   

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Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. HBSS/HEPES for ROS measurement in PMN. protocols.io https://dx.doi.org/10.17504/protocols.io.bhdcj22w Copy   

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Authors: Sven Reischauer
Summary: Zebrafish are an increasingly popular model system to better understand vertebrate physiology. Physical challenge by exercise has become popular to assess physical fitness of genetically manipulated animals or to identify gene regulatory networks that prepare the organism for higher physical demands. The increasing popularity of the zebrafish model system to address questions surrounding physical fitness and physiology has led to the development and commercial availability of sophisticated exercise setups (e.g. swim tunnel respirometers, Loligo® Systems, Viborg, Denmark). However, in many cases, these commercial systems are not available to researchers as they are costly.This protocol describes a simple and effective way to exercise zebrafish. The setup is assembled from components that are present in virtually any laboratory. Its adoptable to constant or increasing endurance exercise as well as interval exercise or sprint tasks. The procedure does not cause stress to the animals as it relies on their natural behavior to swim against water currents to hold their position relative to their visual environment.

Proper citation: Sven Reischauer 2018. Assessing vertebrate physiology: A simple and cost-effective method to exercise adult zebrafish.. protocols.io https://dx.doi.org/10.17504/protocols.io.sa2eage Copy   

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Authors: Daniel Marchal

Proper citation: Daniel Marchal 2018. Preparation of glycerol stocks for V. natriegens. protocols.io https://dx.doi.org/10.17504/protocols.io.uy2exye Copy   

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Authors: Elisa Granato
Group: Elisa Granato

Proper citation: Elisa Granato 2020. Quick'n'Dirty electrocompetent E. coli cells . protocols.io https://dx.doi.org/10.17504/protocols.io.bjpykmpw Copy   

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Authors: Adolfo Maria TAMBELLA, Anna Rita ATTILI
Summary: Objective To determine whether the topical application of platelet-rich plasma (PRP) promotes healing in experimentally-induced full-thickness skin wounds in animals, a systematic review and meta-analysis will be performed.MethodsWeb of Science, Cochrane Library, PubMed, Research Gate, Cochrane Wounds Group, and Veterinary Information Network will be searched to identify randomised and not randomised controlled clinical trials comparing PRP with placebo or with other treatments in animals. Primary outcome: reduction of open wound area; secondary outcomes: healing time and number of healed cases. Effect sizes: Hedges’ g; odds ratio. This protocol was used in the following publication:https://doi.org/10.1371/journal.pone.0191093Tambella AM, Attili AR, Dupré G, Cantalamessa A, Martin S, Cuteri V, Marcazzan S, Del Fabbro M (2018) Platelet-rich plasma to treat experimentally-induced skin wounds in animals: A systematic review and meta-analysis. PLoS ONE 13(1): e0191093. doi: 10.1371/journal.pone.0191093

Proper citation: Adolfo Maria TAMBELLA, Anna Rita ATTILI 2018. Platelet-rich plasma application in experimentally-induced skin wounds in animals: protocol for a systematic review and meta-analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.mnrc5d6 Copy   

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Authors: Monserrat López-Sanmartín, Gaetano Catanese, Amalia Grau, Jose Maria Valencia, JRafa García-March, JINavas
Summary: Real time PCR to identify Haplosporidium pinnae from Pina nobilis DNA.

Proper citation: Monserrat López-Sanmartín, Gaetano Catanese, Amalia Grau, Jose Maria Valencia, JRafa García-March, JINavas 2019. qPCR Identification Haplosporidium pinnae protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.xmyfk7w Copy   

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Authors: Bo-jian Chen, Kai Liu, Lin-jun Zhou, Guilherme Gomes-Silva, Carolin Sommer-Trembo, Martin Plath
Summary: In this protocol, we used computer animated stimuli for two mate choice tests of Gambusia affinis, such that stimulus pairs differed only by (a) body size and (b) locomotor activity, but not in other morphological or behavioural traits that could affect mate choice decisions.

Proper citation: Bo-jian Chen, Kai Liu, Lin-jun Zhou, Guilherme Gomes-Silva, Carolin Sommer-Trembo, Martin Plath 2018. A computer animation based mating preference assessment protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.m67c9hn Copy   

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Authors: Aleksandra Kozyczkowska
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology

Proper citation: Aleksandra Kozyczkowska 2018. Stable tranfection of unicellular relative of animals, Corallochytrium limacisporum, using Lonza Nucleofector. protocols.io https://dx.doi.org/10.17504/protocols.io.r5ud86w Copy   

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Authors: Kouland Thin, Benjamin Johns
Summary: In Cambodia, HIV/AIDS and TB have historically been financed by donors, the Ministry of Health, andhouseholds (via out-of-pocket spending). Donor funding for HIV/AIDS and TB is declining, thegovernment is struggling to generate new resources dedicated to the prevention or treatment of thesediseases. However, in recent years Health Equity Funds (HEFs) have begun to provide financing forHIV/AIDS and TB services. Upcoming social health protection reforms increase the likelihood that newsocial health insurers, e.g. the National Social Security Fund (NSSF), will also cover HIV/AIDS and TBservices, as has been done in other countries in the region, including Vietnam, Thailand, and thePhilippines. The NSSF currently does not clearly specify that HIV/AIDS and TB services are included in thebenefit package; rather the NSSF health benefit package specifies that it covers ‘treatment and careservices with medical professional techniques’.The purpose of this study will be to understand the unit costs of delivering HIV/AIDS and TB services,identify the major components of costs, sources of funding, and to inform HEF (and eventually NSSF)reimbursement rates. In the short term, the results will be used to build an actuarial cost and financingmodel projecting resource needs and likely sources of funding for HIV/AIDS and TB services over 5 years.

Proper citation: Kouland Thin, Benjamin Johns 2019. Estimating the cost of HIV/AIDS and TB services at health facilities in Cambodia. protocols.io https://dx.doi.org/10.17504/protocols.io.2eggbbw Copy   

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Authors: Gunnar Neumann, Aida Andrades Valtuena, James Fellows Yates, Raphaela Stahl, Guido Brandt
Group: MPI-SHH Archaeogenetics
Summary: This protocol describes how to obtain powder from the inner pulp chamber of teeth for the extraction of ancient DNA. It is ideal for the simultaneous isolation of both host endogenous and microbial DNA (e.g. for pathogens).

Proper citation: Gunnar Neumann, Aida Andrades Valtuena, James Fellows Yates, Raphaela Stahl, Guido Brandt 2020. Tooth Sampling from the inner pulp chamber for ancient DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bakqicvw Copy   

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Authors: Neilier Junior
Summary: A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.

Proper citation: Neilier Junior 2020. Hydrochloric Acid-Potassium Chloride Buffer (HCl-KCl). protocols.io https://dx.doi.org/10.17504/protocols.io.bfycjpsw Copy   

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Authors: luanaborba
Group: L. P. Borba-Santos

Proper citation: luanaborba 2020. Screening of compounds for inhibition of Sporothix spp. growth. protocols.io https://dx.doi.org/10.17504/protocols.io.bje9kjh6 Copy   

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Authors: Sebastian Bassi
Summary: Some advices regarding writing Selenium tests. The examples are in Python but the rules are language agnostic, so it can be implemented in any supported language.

Proper citation: Sebastian Bassi 2019. 10 simple rules for writing Selenium automated tests. protocols.io https://dx.doi.org/10.17504/protocols.io.7bbhiin Copy   

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