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Authors: John Rutledge
Group: Mouse Metabolic Phenotyping Centers
Summary: Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) is used to assess blood-brain barrier permeability in vivo in rodent models. Post-processing image analysis is done using our in-house matlab script with patlak linearized regression mathematical modeling.

Proper citation: John Rutledge 2019. UC Davis - Dynamic contrast enhanced magnetic resonance imaging. protocols.io dx.doi.org/10.17504/protocols.io.yh9ft96 Copy   

•   Source: Protocols.io

Authors: Samuel Mehr
Summary: [Imported from this thread.]We are writing a lab handbook, so I spent my afternoon reading 25 lab handbooks to figure out all the things that people put in lab handbooks. Here they are so that you don't have to read 25 lab handbooks. (This is not exhaustive and many of the topics overlap, obviously.)----------------------------------------MISSION STATEMENT. research goals; philosophy of how the lab works; chronological and academic history of the lab; ideas about future projects; why we do what we doROLES & EXPECTATIONS.who's who and what they do all day, including the PI; expectations of everybody from everyone else's perspectiveOPEN SCIENCE.why and how the lab deals with sharing data, code, materials; how the lab does version control; how to do this stuff with lab collaboratorsCOMMUNICATION. how people in the lab talk to each other; things like "always use Slack", "phone calls are only for emergencies"; how long to wait before nudging the PI on something you need; how meetings with PI work and what to prepare for them; check-ins/stand-ups/huddlesLOGISTICS.when and for how long people work; policy on remote work; vacation; where the lab is located and how to get there (especially how to tell *others* how to get there); how to book a conference room; what to do if you get locked outINTERNAL RESOURCES. these are usually a long list of things people in the lab need access to: servers, software packages, commonly used web tools, shared credentials, room keys and other physical resources, etcEXTERNAL RESOURCES.how to get a library card; which building has the best photocopier; useful websites and tutorials, etcONBOARDING. master list of everything that new lab members need to deal with when starting out, including credentials, software, hardware, keys, university ID, etcRECURRING EVENTS.logistics and structure of lab meetings, departmental seminars, etc; daily and weekly task lists (differing by lab roles)DAY-TO-DAY.dress code & hygiene; can you bring your dog to the lab; person X has a peanut allergy so please pack your lunch carefully; health stuff; work-life balanceBEHAVIOR.official code of conduct; policies on scientific integrity, sexual harassment, discrimination; lab culture stuff "the kind of lab we are trying to be" and "things about our lab students like/dislike"; what to do if you have a problem; university-level informationETHICS AND SAFETY.irb procedures; safety procedures; what to do when something goes wrong; what to do/who to call in an emergencyENGAGEMENT.how lab website and social media work; expectations surrounding public outreach, including from official lab accounts; social media policy for personal accounts; protocol for recruiting participants in the community; being a good departmental citizenHOW TO RUN EXPERIMENTS. detailed instructions for doing research; some labs do this for all experiments separately while others have a general set of instructions like "how you should interact with participants"; how to compensate participantsDATA ANALYSIS.how the lab does analyses; expectations concerning how code is written, version controlled, and archived; how to handle data protection and security. this one overlaps with OPEN SCIENCE quite a bitPUBLICATIONS.how the lab deals with authorship, including the differences between listing in acknowledgments & co-authors; checklist of everything to do before a paper is published; policies on preprints, postprints, and open access; preferences about journalsCONFERENCES. how to give a talk; which conferences do lab members usually attend and why; technology considerations surrounding visualizations; data considerations for work-in-progress; discussion of #betterposter or #worseposter, etcMONEY.what grants pay for the lab and why; what the lab will and won't pay for, usually (differs by roles); what grants people could/should apply for; what to expect for conference expenses, computers, etcOFFBOARDING.what to do when you leave the lab; making sure all data & materials are archived properly; transferring credentials; how to stay in touch (what happens to your Slack account)MENTORSHIP AND DEVELOPMENT. how to choose a project; how to get feedback from others on new ideas; how to initiate collaborations inside/outside the lab; doing a thesis/dissertation; professional development at university level; rec lettersWHAT TO DO IF YOU NEED HELP.how to get support from inside or outside the lab; schedule of routine training for new members; things that bear repeating like "always ask questions!"; how to make mistakes productivelyREADING LIST. a list of papers, blog posts, and news articles that all students and lab members should read------------------OK that should about cover it but if I missed stuff please post below. In terms of platforms, I saw handbooks hosted in standalone files (word, pdf, rmd), gdocs, text on github, OSF wikis, gitbooks, and more. (we're using gitbook for now!) time for a cup of tea, bye

Proper citation: Samuel Mehr 2019. Lab Handbook Template. protocols.io dx.doi.org/10.17504/protocols.io.53mg8k6 Copy   

•   Source: Protocols.io

Authors: Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Daniel Richter, Nov 29, 2011based on RNAqueous May 29, 2008 protocol revision C, TURBO DNA-free June 9, 2009 protocol 1907M revision F

Proper citation: Daniel Richter 2017. RNAqueous with TURBO DNA-free and SUPERase-In. protocols.io dx.doi.org/10.17504/protocols.io.iqzcdx6 Copy   

•   Source: Protocols.io

Authors: Bulent Arman Aksoy, Pınar Aksoy, Megan Wyatt, Chrystal M. Paulos, Jeff Hammerbacher
Group: Hammer Lab

Proper citation: Bulent Arman Aksoy, Pınar Aksoy, Megan Wyatt, Chrystal M. Paulos, Jeff Hammerbacher 2019. Human primary T cell culture media. protocols.io dx.doi.org/10.17504/protocols.io.7yihpue Copy   

•   Source: Protocols.io

Authors: Benedikt Fasel, Jörg Spörri, Josef Kröll, Kamiar Aminian
Summary: Calibration procedure for functionally aligning inertial sensors fixed to the shanks, thighs, trunk (e.g. sacrum, sternum), head, arms, and forearms / wrists. The movements were originally designed for analyzing skiing movements where the person is wearing ski boots. However, the same protocol can be applyied also barefoot or with regular shoes.Please refer to the protocol's guidelines for more information how to obtain a sample data set and the matlab code needed to process the functional calibration.

Proper citation: Benedikt Fasel, Jörg Spörri, Josef Kröll, Kamiar Aminian 2017. Functional calibration for trunk, lower- and upper-limb fixed inertial sensors. protocols.io dx.doi.org/10.17504/protocols.io.jzncp5e Copy   

•   Source: Protocols.io

Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. Diabetic Complication:

Proper citation: Eva Feldman 2019. Biochemical Measures of Neuropathy - Western Blot Stripping. protocols.io dx.doi.org/10.17504/protocols.io.3s3gngn Copy   

•   Source: Protocols.io

Authors: Judy Northill, Alyssa Pyke, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time RT-PCR targeting the 5' untranslated region of Yellow fever virus. This protocol was designed and developed at this laboratory.

Proper citation: Judy Northill, Alyssa Pyke, Ian Mackay 2018. Yellow fever virus real-time RT-PCR. protocols.io dx.doi.org/10.17504/protocols.io.rszd6f6 Copy   

•   Source: Protocols.io

Authors: Juliana Ferreira da Silva, Maria Angélica Oliveira, Raylane Ribeiro Anunciação, Eduardo Pereira da Silva, Rodrigo Paidano Alves, Adriano Luis Schunemann, Filipe de Carvalho Victoria, Margéli Pereira de Albuquerque, Antonio Batista Pereira
Summary: The establishment of diatom communities depends on environmental factors such as the type of substrate and geographic conditions that influence the dispersal processes of these organisms. The main goal of this study was to evaluate the similarity between diatom communities associated with the macroalgae Prasiola crispa(Lightfoot) Kützing in relation to spatial distance from six sampled sites located in the South Shetland Islands, Maritime Antarctica. The diatom flora associated with Prasiola crispawas represented by 23 species distributed in 15 genera. Pinnularia australoschoenfelderiZidarova, Kopalová & Van de Vijver, LuticolaaustroatlanticaVan de Vijver, Kopalová, S.A.Spaulding & Esposito, Luticolaamoena Van der Vijver, Kopalová, Zidarova & Levkov, Pinnularia austroshetlandica(Carlson) Cleve-Euler and Psammothidium papilio(D.E. Kellogg et al.) Kopalová & Zidarova were the most abundant species in our samples, which together represented 68% of the total number of individuals collected. There is a great similarity in composition and abundance of the diatom community among the sampled points., which resulted in the absence of a linear pattern of relationship with the distance between sampling points. We conclude that distance was not a factor of differentiation of Antarctic diatom communities associated with terrestrial green macroalgae. Suggesting that Antarctic environments may have unique characteristics with homogeneous abiotic factors, at least related to this substrate.

Proper citation: Juliana Ferreira da Silva, Maria Angélica Oliveira, Raylane Ribeiro Anunciação, Eduardo Pereira da Silva, Rodrigo Paidano Alves, Adriano Luis Schunemann, Filipe de Carvalho Victoria, Margéli Pereira de Albuquerque, Antonio Batista Pereira 2019. Distribution of aerophilous diatom communities associated with terrestrial green macroalgae in the South Shetland Islands, Maritime Antarctica - PROTOCOL. protocols.io dx.doi.org/10.17504/protocols.io.6tcheiw Copy   

•   Source: Protocols.io

Authors: Heather Robeson, Jing Jin, Mohammed S. Orloff
Summary: The protocol is based on the abcam Indirect ELISA protocol.

Proper citation: Heather Robeson, Jing Jin, Mohammed S. Orloff 2020. Indirect ELISA protocol (abcam). protocols.io dx.doi.org/10.17504/protocols.io.bn8umhww Copy   

•   Source: Protocols.io

Authors: Erin Cutts, Alessandro Vannini
Summary: The biGBac system presented by Weissmann et al. (Weissmann et al, 2016 and Weissmann et al, 2018) is a modular insect cell expression system designed for expression of multi-subunit complexes. The system is very powerful, allowing up to 25 genes to be assembled into one baculo-virus expression vector, but does require a reasonable amount of cloning experience in order to attain clones in the 6 day time frame stated in the paper. This guide was written to provide a detailed explanation how the system is designed, how it can be further exploited and provide in-depth practical information that even a novice cloner can follow.

Proper citation: Erin Cutts, Alessandro Vannini 2018. Troubleshooting biGBac: a practical guide. protocols.io dx.doi.org/10.17504/protocols.io.q3ydypw Copy   

•   Source: Protocols.io

Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:Target cells are depleted by incubating the sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads (Cat. No.480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc. Note: This protocol has been optimized to remove washing steps after antibody cocktail and nanobeads incubations, resulting in a shorter and more convenient protocol. This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Selection Kits Protocol - 1. protocols.io dx.doi.org/10.17504/protocols.io.7yxhpxn Copy   

•   Source: Protocols.io

Authors: Mark Dewitt, Julia Wong
Group: Innovative Genomics Institute, CornLab
Summary: In vitro T7 template synthesis and transcriptionsgRNA protocol with SPRI beads

Proper citation: Mark Dewitt, Julia Wong 2015. In vitro transcription of guide RNAs. protocols.io dx.doi.org/10.17504/protocols.io.d4w8xd Copy   

•   Source: Protocols.io

Authors: Bernardo Mainou
Summary: Indirect immunofluorescence infectivity assay for reovirus using LICOR Imaging System

Proper citation: Bernardo Mainou 2017. Fluorescent Focus Unit Assay using LICOR Imaging System. protocols.io dx.doi.org/10.17504/protocols.io.k8fcztn Copy   

•   Source: Protocols.io

Authors: New England Biolabs
Group: New England Biolabs (NEB)

Proper citation: New England Biolabs 2015. E1202 Ligation Mixture. protocols.io dx.doi.org/10.17504/protocols.io.cp6vrd Copy   

•   Source: Protocols.io

Authors: yliu
Group: OnsiteGene 1, XPRIZE Rapid Covid Testing
Summary: This OnsiteGene protocol is designed for testing the nasal swab samples without nucleic acid extraction. It uses the Star Array® Hi-SenseTM COVID-19 Molecular Testing Kit 1.0 in the one-step real-time RT-qPCR test assay to qualitatively detect RNA from SARS-CoV-2 virus in the human respiratory specimen. It combines the reverse transcription technology and real-time PCR method to provide accurate detection of the SARS-CoV-2 coronavirus. Along with the Star Array® SATM Direct Extract Buffer, the protocol supports direct amplification without the need of specific RNA extraction equipment and kit. It significantly reduces the loss of RNA from the sample extraction and purification process, saves time and workload from sample preparation, and minimized the burdens on supply chain. The triplex fluorescence design of the kit simultaneously detects the N1 and Orf1ab genes of the virus and the human RNase P gene as an internal control to ensure the sample quality. This protocol uses the Star Array® XDiveTM Superfast Real-Time RT-qPCR instrument to perform 40-cycle PCR in 8 minutes, and can test up to 16 samples or controls in each run. The total protocol time from sample collection to data interpretation is less than 11 minutes.

Proper citation: yliu 2020. OnsiteGene 1 Protocol Nasal Direct. protocols.io dx.doi.org/10.17504/protocols.io.bkudkws6 Copy   

•   Source: Protocols.io

Authors: Leah Reznikov
Group: SPARC
Summary: RNA Sequencing of Porine Ganglia and brainstem Animals. A total of 44 piglets (Yorkshire Landrace breed, 2–3 days of age) were fed commercial milk replacer (Soweena Litter Life) and allowed a 36–48-h acclimation period before interventions began. The University of Florida Animal Care and Use Committee approved all procedures. Procedures were completed in accordance with federal policies and guidelines. A total of 12 piglets were reserved for RNA sequencing experiments (6 male and 6 female). Airway instillation. After acclimation, piglets were anesthetized with 8% SevoThesia (Henry Schein). The piglets’ airways were accessed with a laryngoscope; a laryngotracheal atomizer (MADgic) was passed directly beyond the vocal folds, as previously described to aerosolize either a 500 μl 0.9% saline control or 1% acetic acid in 0.9% saline solution to the airway. This procedure results in widespread distribution of aerosolized solutions throughout the piglet airway, including the lung. Consistent with acid as a cough-evoking stimulus acetic acid induced cough in 90% or greater of the piglets. Nodose ganglia and brain stem isolation. Forty-eight hours after instillations, piglets were euthanized with a 90 mg/kg intravenous Euthasol solution (Henry Schein). Nodose ganglia were carefully dissected using methods that we previously developed. Ganglia were removed, placed in TRIzol, and stored at −80°C until RNA extraction. Any connective tissues, inflammatory cells, satellite cells, or blood cells covering the ganglia were not removed. A region of the brain stem encompassing the nucleus tractus solitarius, nucleus ambiguous, and dorsal motor nucleus was removed. Briefly, a blunt scissor was used to cut through the sagittal suture of the skull post mortem, and the brain was delicately excised. The cerebellum was removed, and the obex was identified. A section of the brain stem encompassing 2 mm rostral and 2 mm caudal to the obex was then removed and placed in RNAlater (Thermo Fisher Scientific). The block was further trimmed to extend 2 mm in depth and 2 mm lateral from the obex, while still submerged in RNAlater. RNA isolation. RNA from thenodose ganglia, and brain stem were isolated using RNeasy Lipid Tissue kit (Qiagen) with optional DNase digestion (Qiagen). RNA concentrations were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA sequencing. RNA concentration was determined on Qubit 2.0 Fluorometer (ThermoFisher/Invitrogen), RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA with 28S/18S > 1 and RNA integrity number (RIN) ≥ 7 were used for RNAseq library construction. RNAseq library were constructed using NEBNext Ultra Directional RNA library prep kit for Illumina (New England Biolabs) following the manufacturer’s recommendations. Briefly, 1,000 ng of total RNA was used for mRNA isolation using NEBNext Ploy(A) mRNA magnetic isolation module (New England Biolabs, cat. no. E7490). RNA library construction was achieved using NEBNext Ultra Directional RNA library prep kit for Illumina (New England Biolabs; cat. no. E7420). Thirty-six barcoded libraries were sized on the bioanalyzer and quantitated by QUBIT. Libraries were pooled in an equimolar ratio and sequenced by Illumina HiSeq 3000 2X100 cycles run for total of six runs (Illumina). RNA library construction was performed at the Interdisciplinary Center for Biotechnology Research (ICBR) Gene Expression & Genotyping Core, University of Florida (UF). HiSeq 3000 sequencing run was performed at the ICBR NextGen DNA Sequencing core, UF. The nodose ganglia and brain stem of three individual piglets for each condition were prepared separately and used. Mapping and differential gene expression. The quality of the RNA-Seq sequence data was first evaluated using FastQC before further downstream analysis. Low-quality sequences were removed, and the poor-quality part of the reads were trimmed using Trimmomatic. Star Aligner was used to map high-quality paired-end reads to Sus scrofa genome, Sscrofa11.1. Expression was obtained using RSEM. The expected read counts and fragments per kilobase of transcript per million-mapped reads (FPKM) were extracted for further analysis. The estimated read counts were taken as input for edgeR to perform differential expression analysis using generalized linear models. The threshold for calling significantly differentially expressed genes was set at FDR 0.05 with the average FPKM for at least one of each comparison group being higher than 0. Additional criteria using P This protocol accompanies the following publication 3. Reznikov LR, Liao YSJ, Gu T, Davis K, Kuan SP, Atanasova KR, Dadural JS, Collins EN, Guevara MV, Vogt KM (2019) Sex-specific airway hyperreactivity and sex-specific transcriptome remodeling in neonatal piglets challenged with intra-airway acid. AJP-Lung. Jan 1;316(1):L131-L143. doi: 10.1152/ajplung.00417.2018. Epub 2018 Nov 8. https://www.physiology.org/doi/full/10.1152/ajplung.00417.2018#

Proper citation: Leah Reznikov 2019. RNA Sequencing of Porine Ganglia and brainstem. protocols.io dx.doi.org/10.17504/protocols.io.w5sfg6e Copy   

•   Source: Protocols.io

Authors: Andreea S
Group: iGEM Groningen 2020
Summary: His tag purification uses the technique of immobilised metal affinity chromatography. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. It has been studied that among amino acids constituting proteins, histidine is strongly involved in the coordinate bond with metal ions. Therefore, if a number of histidines are added to the end of the protein by genetic engineering, the affinity of the protein for the metal ion is remarkably increased and the basic idea is that purification can be easily carried out. When a protein having a His tag is brought into contact with a carrier on which a metal ion such as nickel is immobilized, the histidine residue chelates the metal ion and binds to the carrier. Since other proteins do not bind to the carrier, they can be washed off with a buffer. Thereafter, it is possible to recover the protein having the His tag with high purity.

Proper citation: Andreea S 2020. His-tag purification . protocols.io dx.doi.org/10.17504/protocols.io.bnw5mfg6 Copy   

•   Source: Protocols.io

Authors: Dr. Steven Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), CyanoHABs
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.Modified from: Watanabe, Makoto M. "Fresh-and salt-water forms of Spirulina platensis in axenic cultures." Bull. Jpn. Soc. Phycol. 25 (1977): 371-377.

Proper citation: Dr. Steven Wilhelm 2017. CT Media. protocols.io dx.doi.org/10.17504/protocols.io.h9kb94w Copy   

•   Source: Protocols.io

Authors: Randie Bundy
Summary: This protocol describes the procedures for making a 1.5 mol L-1 nitrite reagent for measuring total dissolved and labile cobalt using cathodic stripping voltammetry.

Proper citation: Randie Bundy 2016. Nitrite reagent for dissolved cobalt analyses. protocols.io dx.doi.org/10.17504/protocols.io.evgbe3w Copy   

•   Source: Protocols.io

Authors: Dania Villarnovo

Proper citation: Dania Villarnovo 2018. Western Blotting Protocol. protocols.io dx.doi.org/10.17504/protocols.io.rdfd23n Copy   

•   Source: Protocols.io