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Authors: Joshua Welsh, Jennifer Jones
Group: Translational Nanobiology Section
Summary: This protocol outlines the steps required to catalogue fluorescence reference materials using the FCMPASS software. This is one of a number of protocols in the pipeline for performing small particle calibration using the fcmpass software package.
Proper citation: Joshua Welsh, Jennifer Jones 2020. FCMPASS - Cataloguing fluorescence reference materials. protocols.io dx.doi.org/10.17504/protocols.io.bhvvj666 Copy
Authors: Steven Burgess
Group: OpenPlant Project
Summary: Instructions to make 50 mL of incubation buffer for protoplast isolation.Adapted from Yoo et al. 2007 http://www.nature.com/nprot/journal/v2/n7/full/nprot.2007.199.html
Proper citation: Steven Burgess 2016. Protoplast Isolation - Incubation Buffer. protocols.io dx.doi.org/10.17504/protocols.io.fd3bi8n Copy
Authors: Diep Ganguly, Timothy Rhodes, Nay Chi Khin, Estee E Tee, Kai Xun Chan
Group: Pogson Genomics Group
Summary: Protocol for recombinant protein expression in E. coli for protein purification and subsequent enzyme assays, protein crystallography etc.
Proper citation: Diep Ganguly, Timothy Rhodes, Nay Chi Khin, Estee E Tee, Kai Xun Chan 2019. Heterologous protein expression in E. coli. protocols.io dx.doi.org/10.17504/protocols.io.95qh85w Copy
Authors: Anastasios Tsaousis
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Anastasios Tsaousis 2017. Transfected Naegleria Fluorescence Microscopy. protocols.io dx.doi.org/10.17504/protocols.io.hpvb5n6 Copy
Authors: Yuan Wen, Ivan Vechetti , Taylor Valentino, John J McCarthy
Summary: Extraction of DNA, RNA, and protein from the same sample would allow for direct comparison of genomic, transcriptomic, and proteomic information. Commercially available kits exhibit poor protein yield and TRIzol® reagent produces a protein pellet that is extremely difficult to solubilize. In response to these limitations, this study presents an optimized method for the extraction of protein from the organic phase of TRIzol® reagent that allows for higher yield recovery of skeletal muscle protein compared to direct homogenization in a common protein lysis buffer. The presented method is inexpensive, simple and fast; requires no additional treatment of the protein pellet for dissolution; and is compatible with downstream western blot applications.
Proper citation: Yuan Wen, Ivan Vechetti , Taylor Valentino, John J McCarthy 2020. High-Yield Skeletal Muscle Protein Recovery from TRIzol® after RNA and DNA Extraction. protocols.io dx.doi.org/10.17504/protocols.io.bii3kcgn Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to prepare 0.9% Saline Solution. 0.9% Sterile Saline solution is used as an initial rinse for the brains of mice prior to perfusion. Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Proper citation: Allen Institute for Brain Science 2020. 0.9% Saline Solution. protocols.io dx.doi.org/10.17504/protocols.io.bg44jyyw Copy
Authors: Rose Jones, Beth Orcutt
Group: Orcutt Deep Biosphere Lab
Summary: The following protocol describes how to prepare microbial fuel cells (MFCs) with Indium Tin Oxide (ITO) electrodes for use as a poised potential enrichment tool, specifically an oxic system to enrich for the cathode-oxidizing community. The protocol describes setting up four “online” MFCs that are connected to a potentiostat at a particular set voltage plus one “offline” unconnected control (to account for non-electrode related changes). One of the online MFCs is a “negative” killed substrate control (to account for abiotic medium-source current fluctuations) and the remaining three MFCs being replicate live samples. This protocol is based on advice primarily from Frauke Kracke and Annette Rowe.
Proper citation: Rose Jones, Beth Orcutt 2019. Bioelectrochemistry protocol For CHI Potentiostat. protocols.io dx.doi.org/10.17504/protocols.io.xihfkb6 Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: The cells targeted by the Streptavidin Nanobeads are either selected or depleted by incubating your sample with the magnetic particles after incubating with a biotin-conjugated antibody or antibody cocktail. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.
Proper citation: Kelsey Miller 2016. MojoSort™ Streptavidin Nanobeads Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e2rbgd6 Copy
Authors: Yaowu Yuan
Group: Mimulus
Summary: This protocol is part of a collection for Mimulus in planta transformation.
Proper citation: Yaowu Yuan 2019. Plant Infiltration for Mimulus in Planta Transformation. protocols.io dx.doi.org/10.17504/protocols.io.3rtgm6n Copy
Authors: James P. O’Callaghan, PhD
Summary: This is the protocol to be used in the quantification of glial fibrillary acidic protein (GFAP) in mouse brain tissue.
Proper citation: James P. O’Callaghan, PhD 2018. GFP ELISA Assay Protocol. protocols.io dx.doi.org/10.17504/protocols.io.hsub6ew Copy
Authors: Adriana Alberti, Julie Poulain, Stefan Engelen, Karine Labadie, Sarah Romac, Isabel Ferrera, Guillaume Albini, Jean-Marc Aury, Caroline Belser, Alexis Bertrand, Corinne Cruaud, Corinne Da Silva, Carole Dossat, Frédéric Gavory, Shahinaz Gas, Julie Guy, Maud Haquelle, E'krame Jacoby, Olivier Jaillon, Arnaud Lemainque, Eric Pelletier, Gaëlle Samson, Marc Wessner, Genoscope Technical Team, Silvia G. Acinas, Marta Royo-Llonch, Francisco M. Cornejo-Castillo, Ramiro Logares, Beatriz Fernández-Gómez, Chris Bowler, Guy Cochrane, Clara Amid, Petra Ten Hoopen, Colomban De Vargas, Nigel Grimsley, Elodie Desgranges, Stefanie Kandels-Lewis, Hiroyuki Ogata, Nicole Poulton, Michael E. Sieracki, Ramunas Stepanauskas, Matthew B. Sullivan, Jennifer R. Brum, Melissa B. Duhaime, Bonnie T. Poulos, Bonnie L. Hurwitz, Stéphane Pesant, Eric Karsenti, Patrick Wincker
Group: Tara Oceans
Summary: This protocol describes the sequencing library preparation for the Tara Oceans expedition and is part of Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition. Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.
Proper citation: Adriana Alberti, Julie Poulain, Stefan Engelen, Karine Labadie, Sarah Romac, Isabel Ferrera, Guillaume Albini, Jean-Marc Aury, Caroline Belser, Alexis Bertrand, Corinne Cruaud, Corinne Da Silva, Carole Dossat, Frédéric Gavory, Shahinaz Gas, Julie Guy, Maud Haquelle, E'krame Jacoby, Olivier Jaillon, Arnaud Lemainque, Eric Pelletier, Gaëlle Samson, Marc Wessner, Genoscope Technical Team, Silvia G. Acinas, Marta Royo-Llonch, Francisco M. Cornejo-Castillo, Ramiro Logares, Beatriz Fernández-Gómez, Chris Bowler, Guy Cochrane, Clara Amid, Petra Ten Hoopen, Colomban De Vargas, Nigel Grimsley, Elodie Desgranges, Stefanie Kandels-Lewis, Hiroyuki Ogata, Nicole Poulton, Michael E. Sieracki, Ramunas Stepanauskas, Matthew B. Sullivan, Jennifer R. Brum, Melissa B. Duhaime, Bonnie T. Poulos, Bonnie L. Hurwitz, Stéphane Pesant, Eric Karsenti, Patrick Wincker 2020. Sequencing library preparation. protocols.io dx.doi.org/10.17504/protocols.io.qwidxce Copy
Authors: Priota Islam
Group: Behavioural Genomics
Summary: Our aim is to study addiction in C elegans with cocaine being the drug choice of abuse. This short experiment is done to check how the worms react instantaneously upon exposure to this drug for the first time.
Proper citation: Priota Islam 2019. Tracking acute response of C elegans to cocaine. protocols.io dx.doi.org/10.17504/protocols.io.xvffn3n Copy
Authors: Alexandra Ehl, David Frommholz, Nadine Stefanczyk
Summary: Purification Guide for the Isolation of Antibodies with ChroDrip Columns by DALEX Biotech.Easy and quick small scale antibody purification from various sources and species.Each ChroDrip column has a binding capacity of > 15 mg/ml (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. ChroDrip - ProteinG. protocols.io dx.doi.org/10.17504/protocols.io.uwbexan Copy
Authors: Igem Dusseldorf
Proper citation: Igem Dusseldorf 2019. Double Digestion of Insert DNA. protocols.io dx.doi.org/10.17504/protocols.io.79ehr3e Copy
Authors: David Eccles
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocol is for comparing two different samples at the transcript level, using long reads that are mapped to transcripts.Input(s): stranded fastq files (see steps 1-8 of Stranded Mapping from Long Reads), transcript reference fasta file, annotation fileOutput(s): transcript table, sorted by differential coverage, annotated with gene name / description / location
Proper citation: David Eccles 2018. Stranded Transcript Count Table Generation from Long Reads. protocols.io dx.doi.org/10.17504/protocols.io.smuec6w Copy
Authors: Sean Sterrenberg, David Gallacher, Melissa Schmidt, Rebecca Rugel, Summer Gerke, Kenneth Gundle, Ryan Wallenberg
Group: Smokerlyzer Study, PVAMC
Summary: Background: Tobacco use is associated with increased post-operative complications and is often underreported by patients. Traditional biochemical testing methods, including serum and urine cotinine assays, do not differentiate active smoking from nicotine replacement therapy (NRT). This study intends to evaluate the effectiveness of a novel point-of-care carbon monoxide (CO) breath test to verify smoking status and differentiate active smoking from NRT pre-operatively in a cohort of veteran orthopaedic patients.
Methods: This is a single-institution, prospective cohort study of adult orthopaedic patients at the Portland VA Medical Center to implement and evaluate a point-of-care CO breath test, the Smokerlyzer® Micro EC50, for pre-operative smoking status verification. Patients who are indicated for orthopaedic surgery in the pre-operative clinic will be offered inclusion. Self-reported smoking status will be obtained pre-operatively to categorize patients as non-smoker, quitters, or active smokers. Exhaled CO and serum cotinine levels will be obtained at the initial clinic visit, pre-operative visit, and day of surgery. Primary endpoints are concordance between self-reported smoking status and CO levels, and concordance between exhaled CO and serum cotinine levels.Discussion: Pre-operative smoking cessation interventions have been shown to decrease smoking rates on the day of surgery and promote abstinence 30 days post-operatively. NRTs are commonly used to promote smoking cessation, however their effect on surgical outcomes are not known. This study aims to validate the use of point-of-care testing that differentiates active smoking from NRT, and identify patients at risk for smoking status misclassification. Results would provide preliminary data to directly test NRT use, separate from tobacco use, and surgical outcomes.Specific Aims: 1. To determine if expired air CO levels can be used to identify patients that are at risk of smoking status misclassification pre-operatively, including patients that are on nicotine replacement therapy. 2. To evaluate the concordance between self-reported smoking and expired air CO levels in the veteran orthopedic population. 3. To evaluate the concordance between pre-operative expired air CO and typical serum levels in the orthopedic population. Hypotheses: 1. Expired air CO testing will allow for differentiation between active smokers and those on nicotine replacement therapy compared to serum cotinine testing. 2. Self-reported “quitters” will have lower agreement between self-reported smoking status and biological testing compared to self-reported “non-smokers” and “active smokers.”3. Compared to serum cotinine testing, expired air CO testing will allow for non-inferior accuracy in verifying smoking status.
Proper citation: Sean Sterrenberg, David Gallacher, Melissa Schmidt, Rebecca Rugel, Summer Gerke, Kenneth Gundle, Ryan Wallenberg 2019. Is Expired Air Carbon Monoxide Testing Effective for Screening of Cigarette Use in Orthopaedic Patients?. protocols.io dx.doi.org/10.17504/protocols.io.7crhiv6 Copy
Authors: Froylan Sosa, Peter Hansen
Summary: This is a simple protocol for collecting skin biopsies from cattle. The procedure described here was used to collect skin for histological analysis but it could also be used to collect skin for PCR or other purposes. Our experience indicates that there are few complications with the procedure. The most common is that occasionally there is a slight amount of bleeding that can be treated by applying pressure using a gauze. Healing occurs rapidly and biopsy sites are difficult to detect 14 days after the procedure. The protocol is best read by using the attached pdf file.
Proper citation: Froylan Sosa, Peter Hansen 2020. Collection of skin biopsies from cattle. protocols.io dx.doi.org/10.17504/protocols.io.bm3yk8pw Copy
Authors: Paloma Varela
Proper citation: Paloma Varela 2019. Holo-ZitRMG binding to dsDNA fragments by ITC. protocols.io dx.doi.org/10.17504/protocols.io.vg5e3y6 Copy
Authors: Stemcell Technologies
Group: STEMCELL Technologies
Summary: Preparing a single cell suspension from a frozen starting sample is critical for optimizing cell isolations by avoiding additional cell loss and enabling the maximum labeling of target cells. Performing cell separation on clumpy cell samples can result in lower recovery and may interfere with proper labeling of the target cells. Samples may sometimes appear "clumpy" when they have been exposed to repeated freeze/thaw cycles or enzymatic tissue dissociation. These cell clumps occur because environmental stresses can accelerate the rate of cell death within the sample, resulting in the release of "sticky" DNA molecules from the dying cells that can clump neighboring cells together. Adding the endonuclease deoxyribonuclease I (DNase I) into your sample can minimize the presence of free-floating DNA fragments and cell clumps. This protocol describes a method to harvest cells and prepare a clump-free single cell suspension from a frozen cell sample prior to performing cell separation.
Proper citation: Stemcell Technologies 2019. How to Prepare a Single Cell Suspension from a Frozen Cell Sample. protocols.io dx.doi.org/10.17504/protocols.io.4umgwu6 Copy
Authors: Lukas Snoek, Tinka Beemsterboer
Group: Spinoza Centre, REC-L
Summary: Run an MRI pilot at Spinoza REC.This protocol will help you running your pilot.
Make sure to read this protocol before coming to the pilot so you know what is expected of you and which choices you will be going to make.
Proper citation: Lukas Snoek, Tinka Beemsterboer 2019. MRI pilot. protocols.io dx.doi.org/10.17504/protocols.io.w8hfht6 Copy
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