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Authors: Dennis Dienst
Group: CyanoWorld

Proper citation: Dennis Dienst 2017. Ethidiumbromide-free RNA sample preparation for agarose gels . protocols.io https://dx.doi.org/10.17504/protocols.io.j3xcqpn Copy   

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Authors: Sarah Hessen-Schmidt

Proper citation: Sarah Hessen-Schmidt 2016. Binding Buffer (3M GuHCl, 3.75M NH4Ac pH 6). protocols.io https://dx.doi.org/10.17504/protocols.io.fjsbkne Copy   

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Authors: jnesvet
Summary: The Stanford University and Purigen Biosystems team's SARS-CoV-2 assay leverages on-chip microfluidics to eliminate laborious and time consuming steps associated with standard molecular diagnostics such as solid phase spin-column extraction and PCR amplification. Purification of nucleic acids from a variety of biological sources is achieved in a one-step, automated fashion using on-chip isotachophoresis (ITP). The purified nucleic acids are then amplified using reverse transcription (RT) loop-mediated isothermal amplification (LAMP) in 30 minutes, less than half of the time associated with standard qPCR. We then use CRISPR-Cas12 fluorescent detection to identify amplicons associated with the SARS-CoV-2 genome for enhanced specificity. 

Proper citation: jnesvet 2021. Stanford and Purigen Biosystems Microfluidics Team protocol XPRIZE_Hudson Alpha. protocols.io https://dx.doi.org/10.17504/protocols.io.brixm4fn Copy   

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Authors: Kalle Pärn, Javier Nunez Fontarnau, Marita A. Isokallio, Timo Sipilä, Elina Kilpelainen, Aarno Palotie, Samuli Ripatti, Priit Palta
Group: FIMM HumGen Sequencing Informatics

Proper citation: Kalle Pärn, Javier Nunez Fontarnau, Marita A. Isokallio, Timo Sipilä, Elina Kilpelainen, Aarno Palotie, Samuli Ripatti, Priit Palta 2019. Genotyping chip data lift-over to reference genome build GRCh38/hg38. protocols.io https://dx.doi.org/10.17504/protocols.io.xbhfij6 Copy   

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Authors: Jessica Sacher

Proper citation: Jessica Sacher 2018. Immunogold labeling of bacterial cells for transmission electron microscopy (TEM). protocols.io https://dx.doi.org/10.17504/protocols.io.mv5c686 Copy   

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Authors: Max Marrone
Group: Opentrons COVID-19 Testing
Summary: Opentrons and the Open Medicine Institute are developing an automated high-throughput COVID-19 testing protocol to submit to the FDA for an Emergency Use Authorization as a diagnostic.The standard assay for this type of infectious disease testing is quantitative PCR (qPCR), and in this case reverse transcriptase qPCR since COVID-19 is an RNA virus. After patient samples are collected in public health facilities, doctors offices, and hospitals, they are sent to the lab for processing, which happens in four steps:Sample IntakeRNA ExtractionqPCR SetupRT-qPCR AssayOpentrons OT-2 robots carry out most of this work. However, human operators are needed for some key tasks, like:Moving samples between stationsPreparing certain reagentsRunning the qPCR machineLogging data

Proper citation: Max Marrone 2020. Opentrons COVID-19 testing (Randox/qPCR common path, stations A & B, 24 samples). protocols.io https://dx.doi.org/10.17504/protocols.io.bemfjc3n Copy   

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Authors: Sade Aisha Folashade John, Patrick E. Akpaka, Chandrashekhar Unakal, Arvind Kurhade, Angel Justiz-Vaillant
Summary: Enterobacteriaceae the leading cause of gastroenteritis. These gram- negative bacteria from species like Escherichia, Shigella, Salmonella, Vibrio and Helicobacter etc are among those that cause severe disease in consumers, especially those that indulge in uncooked seafood. Oysters have been shown to harbour these pathogenic organisms, which pose a major health risk. Numerous outbreaks of gastroenteritis due to oyster consumption have been reported worldwide because of the enteric bacteria as well as viral pathogen contaminationThis research was conducted to identify the Enterobacteriaceae present in oysters sold by vendors in Trinidad. During a five-month period (the rainy season, May - September), a total of 156 oyster samples (comprising 104 oysters samples and 52 water samples of the prepared, unprepared oyster cocktails plus the water used by vendors) were analysed. These samples were collected from roadside vendors operating near to the coastal line of the Western part of Trinidad These were processed at the microbiology unit of the Department of Para-clinical Sciences of the University of the West Indies, St. Augustine using standard microbiology techniques. The antibiotic susceptibility profiles of the recovered organisms were performed using the Kirby Bauer method for the following antibiotics- Tetracycline, Ampicillin, Trimethoprim-Sulfamethoxazole, Cefuroxime and Ceftazidime. The SPSS 21 programme was used to analyse the biometric data and a chi-square test was used to determine if there was any significant difference between the kinds of preparation cocktails with respect to the coliform and organisms found.Results of the 104 oyster samples tested, 112 isolates were obtained, 13.4% (15/112) were E.coli of which 0.1% (1/15) was the 0157:H7 strain; 72.3% (81/112) were Shigella species of which 27.2% (22/81) were Shigella dysenteriae; 14.3% (16/112) were Salmonella species of which 25% (4/16) were Salmonella cubana. The susceptibility profiles of the organisms revealed that 86.5% of Shigella, 100% of E.coli and 93.7% of the Salmonella were resistant to multiple antibiotics. With the recovery of these organisms from these samples, the health of the Trinidadian and foreign consumers of mangrove oysters is at risk particularly from Shigella, E.coli and Salmonella species. As a result, there should be an increased need for public awareness as well as regulations by the Ministry of Health to be made so that illness from these hazardous Enterobacteriaceae can be prevented.

Proper citation: Sade Aisha Folashade John, Patrick E. Akpaka, Chandrashekhar Unakal, Arvind Kurhade, Angel Justiz-Vaillant 2020. Copy of Detection of total and faecal coliforms in the oysters. protocols.io https://dx.doi.org/10.17504/protocols.io.bj6gkrbw Copy   

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Authors: Michael Eadon, Daria Barwinska, Ying-Hua Cheng, Michael J. Ferkowicz, Samir V. Parikh, Brad H. Rovin, John P. Shapiro, Pierre C. Dagher, Tarek M. El-Achkar
Group: KPMP
Summary: Recent advances in multi-scale interrogation of human tissue, including advanced imaging techniques and the powerful application of large dataset “omics”, have displayed significant promise toward identifying new and specific therapeutic targets, predicting disease progression, and individualizing treatment in participants with acute and chronic kidney disease. Our site, formed by the alliance of Indiana University and Ohio State University, will implement an unbiased tissue interrogation workflow for KPMP human kidney biopsies that integrates largescale 3D tissue imaging for quantitative supervised and unsupervised analysis/cytometry with sub-segmental “omics” data on the same kidney biopsy specimen. The sub-segmental “omics” pipeline will use fluorescence based Laser MicroDissection (LMD) to isolate specific nephron segments and interstitial/other targeted areas, for downstream analysis with transcriptomics and proteomics. The omics analysis will be eventually expanded to include bulk epigenetics. Acting harmoniously with other KPMP sites, our interrogation techniques are expected to facilitate back-mapping of key molecular pathways to the biopsy, which can subsequently identify foci of injury and/or regeneration that can undergo targeted sampling to generate further enriched omics. Therefore, in addition to a significant contribution to the human kidney atlas, our approach will complement other interrogation techniques within KPMP by providing tissue context and increasing spatial resolution for molecular signatures that arise in heterogeneous areas during kidney disease.

Proper citation: Michael Eadon, Daria Barwinska, Ying-Hua Cheng, Michael J. Ferkowicz, Samir V. Parikh, Brad H. Rovin, John P. Shapiro, Pierre C. Dagher, Tarek M. El-Achkar 2020. Laser microdissection for regional transcriptomics and proteomics. protocols.io https://dx.doi.org/10.17504/protocols.io.8rkhv4w Copy   

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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2020. MojoSort™ Whole Blood Human CD8 Nanobeads Whole Blood Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bbujinun Copy   

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Authors: Merissa Chen, Nina Draeger, Martin Kampmann, Kun Leng, Emmy Li, Connor Ludwig, Gregory Mohl, Avi Samelson, Sydney Sattler, Ruilin Tian
Group: Neurodegeneration Method Development Community, KampmannLab
Summary: This protocol describes thedifferentiation of iPSCs with stably integrated doxycycline-inducible Ngn2 (such as i3Ns).

Proper citation: Merissa Chen, Nina Draeger, Martin Kampmann, Kun Leng, Emmy Li, Connor Ludwig, Gregory Mohl, Avi Samelson, Sydney Sattler, Ruilin Tian 2019. iNeuron pre-differentiation & differentiation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8dths6n Copy   

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Authors: Else Marie Bartels
Summary: Complement activation correlates to rheumatoid arthritis disease activity, and increased amounts of the complement split product C5a is observed in synovial fluids from rheumatoid arthritis patients. Blockade of C5a or its receptor (C5aR) is efficacious in several arthritis models. The aim of this study is to investigate the role of C5a and C5aR in human rheumatoid arthritis and psoriatic arthritis – both with respect to expression and function.Synovial fluid, blood and synovial samples will be obtained from rheumatoid arthritis, psoriatic arthritis and osteoarthritis patients as a less inflammatory arthritis type, and blood from healthy subjects. Cells infiltrating synovial tissue will analysed by immunohistochemistry and flow cytometry. SF and blood will be analysed for biomarkers by flow cytometry or ELISA. The effect of a blocking anti-human C5aR mAb on leukocyte migration will be determined using a Boyden chamber. Appropriate statistical tests will be applied for comparisons.

Proper citation: Else Marie Bartels 2018. The role of C5a in Rheumatoid and Psoriatic Arthritis in comparison with Osteoarthritis - Profiling of Synovial and Systemic Biomarkers. protocols.io https://dx.doi.org/10.17504/protocols.io.kmxcu7n Copy   

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Authors: Kenneth Schackart, Kattika Kaarj
Group: 481b Laboratory
Summary: This procedure details how to culture E. coli K-12 in suspension and quantify cultured bacteria using a spectrometer.

Proper citation: Kenneth Schackart, Kattika Kaarj 2019. E. coli Optical Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.xu3fnyn Copy   

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Authors: Milos Kalc, Ramona Ritzmann, Vojko Strojnik
Summary: Purpose: The aim of the study was to investigate the origin and magnitude of neuromuscular fatigue induced by half-squat whole-body vibration.Methods: Ten young, recreationally trained adults participated in six fatiguing protocols, each consisting of several sets of 30, 60 or 180 s static squatting superimposed with vibration (WBV30, WBV60, WBV180) or without vibration (SHAM30, SHAM60, SHAM180) for a total exercise exposure of 9-minutes in each trial. Maximum voluntary contraction (MVC), level of voluntary activation (%VA), single twitch peak torque (TWPT), low- (T20) and high-frequency (T100) doublets, and low-to-high-frequency fatigue ratio (T20/100) were assessed before, immediately after, 15 and 30 minutes after each fatiguing protocol.

Proper citation: Milos Kalc, Ramona Ritzmann, Vojko Strojnik 2020. Effects of Whole-Body Vibrations on Neuromuscular Fatigue. protocols.io https://dx.doi.org/10.17504/protocols.io.beadjaa6 Copy   

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Authors: Mike Edwards
Group: Mike J. Edwards
Summary: By means of the density functional theory framework I tackle the long-standing problem of a polymer star interpenetrating with a polymer brush at thermal equilibrium. Remarkably, the star is repelled to the outside of the brush once it sucks into the brush. It turns out that there could be a highly fluctuating region at the brush edge. The highly fluctuating region would be responsible for discontinuous absoption transitions by brushes. However, up to an small interpenetration length, below which asphericity of the star is maintained, the star gets collapsed by sucking more and more into the brush.

Proper citation: Mike Edwards 2021. Interpenetration-between-a-polymer-star-and-a-polymer-brush. protocols.io https://dx.doi.org/10.17504/protocols.io.bsmxnc7n Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol is used to dissociate adult (10 week) CD-1 mouse liver "on ice", preserving authentic gene expression profiles. The first layer consists of 3.5 hours rotation at 4 °C in 0.25% trypsin, and the second layer consists of 15 min. in bacillus licheniformis, with trituration on ice. The yield is 5000 cells/mg with 97% viability (as measured by trypan blue). 

Proper citation: Andrew Potter 2018. Adult mouse liver dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.r8id9ue Copy   

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Authors: Liz O'Brien, Connor Tsuchida
Group: The Center for Genome Editing and Recording
Summary: This protocol describes a CasX Cleavage Assay.

Proper citation: Liz O'Brien, Connor Tsuchida 2019. CasX Cleavage Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.8wshxee Copy   

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Authors: Alan J. Cone
Group: Ju Lab
Summary: Separates molecules based on size. Great for checking DNA after a Restriction Digest.

Proper citation: Alan J. Cone 2015. Gel Electrophoresis. protocols.io https://dx.doi.org/10.17504/protocols.io.dbd2i5 Copy   

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Authors: Mel Duhaime
Group: VERVE Net, Sullivan Lab

Proper citation: Mel Duhaime 2016. PSS2 PCR Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.dfm3k5 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. T cells produce IL-14, and its principal effects are stimulation of activated B cell proliferation and inhibition of immunoglobulin secretion. [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-14 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj3dkqi6 Copy   

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Authors: J. Frederick
Summary: Peptide purification counts after the synthesis, or other forms of making of it. For drug applied peptide like cell-penetrating peptides are supposed to ba paid speical attention as impurities may bring unwanted effects, as well as APIs peptides like pt-141.

Proper citation: J. Frederick 2016. HPLC Purification of Peptides. protocols.io https://dx.doi.org/10.17504/protocols.io.gvsbw6e Copy   

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