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Authors: Benjamin Emert
Group: RajLab
Summary: Protocol for preparing NGFR-mNG2 tagged WM989 A6-G3 melanoma cells for sorting.
Proper citation: Benjamin Emert 2018. NGFR-mNG2 sorting protocol. protocols.io dx.doi.org/10.17504/protocols.io.rh6d39e Copy
Authors: Skye Waterland, Yang Li
Group: Dr. Shen's Lab Group
Summary: Create single gRNA vectors for targeted cloning utilizing CRISPR or CRISPR-based systems.
Proper citation: Skye Waterland, Yang Li 2020. Target Guide Sequence Cloning Protocol . protocols.io dx.doi.org/10.17504/protocols.io.bij2kcqe Copy
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 and less antibody cocktail that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:2 to 1:10 for the antibody cocktail can be used. Dilutions ranging from 1:5 to 1:20 for the Streptavidin Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.
Proper citation: Sam Li 2019. MojoSort™ Isolation Kits Column Protocol - 3. protocols.io dx.doi.org/10.17504/protocols.io.7behije Copy
Authors: Eric Danner
Summary: This is the Tn5 gDNA prep published in the supplementary file of the UDiTaS paper (https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4561-9).Usage: measuring chromosome fusions, InDels, mRNA splicing, structural variants, large resection, off target integration. -> Some of these DNA repair outcomes are demonstrated using such a sample preparation in this paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455975/ ). My protocol is a modified version of UDiTaS but with a few tweaks like using nested primers. You will need a protein facility to produce the recombinant Tn5.Software for analysis is on https://github.com/ericdanneror https://github.com/editasmedicine/uditas
Proper citation: Eric Danner 2020. Tn5 Library Prep for Deep Sequencing Loci of CRISPR/Cas9 Edited Cells. Single gene specific primer amplification (UDiTaS protocol with alterations). protocols.io dx.doi.org/10.17504/protocols.io.7k2hkye Copy
Authors: Abdul Obeid, Peter WIttek, Peter D. Bruza
Proper citation: Abdul Obeid, Peter WIttek, Peter D. Bruza 2018. Evaluating probabilistic programming languages for simulating quantum correlations. protocols.io dx.doi.org/10.17504/protocols.io.u79ezr6 Copy
Authors: Ken Youens-Clark
Group: MetaFunc Course
Proper citation: Ken Youens-Clark 2016. Setting $PATH on UA HPC. protocols.io dx.doi.org/10.17504/protocols.io.fribm4e Copy
Authors: Chenhui Chang
Proper citation: Chenhui Chang 2017. Extraction of PLFA from wood debris. protocols.io dx.doi.org/10.17504/protocols.io.ixmcfk6 Copy
Authors: G Jason Smith
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Environmental Biotechnology Lab
Summary: This is a modification of the origianl Chelex 100 extraction described by Walsh, Metzger and Higuchi (1991 BioTechniques 10(4):506). Adding PVPP facilitates working with environmental samples as well as pure cultures hi in phenolics or other contaminants.
Proper citation: G Jason Smith 2017. Simple DNA Extraction for Phytoplankton Using Chelex 100. protocols.io dx.doi.org/10.17504/protocols.io.h58b89w Copy
Authors: Benoit Besson, Florian Sonthonnax, Magalie Duchateau, Youcef Ben Khalifa, Florence Larrous, Hyeju Eun, Véronique Hourdel, Mariette Matondo, Julia Chamot-Rooke, Regis Grailhe, Hervé Bourhy
Proper citation: Benoit Besson, Florian Sonthonnax, Magalie Duchateau, Youcef Ben Khalifa, Florence Larrous, Hyeju Eun, Véronique Hourdel, Mariette Matondo, Julia Chamot-Rooke, Regis Grailhe, Hervé Bourhy 2017. Bioluminescence Resonance Energy Transfer at various ratios of expressed proteins. protocols.io dx.doi.org/10.17504/protocols.io.jepcjdn Copy
Authors: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Rizwan Ansari, Fiona Behan, Mathew Garnett
Group: Cellular Generation and Phenotyping
Summary: This protocol is used to identify the optimum blasticidin concentration for the selection of Cas9 positive cancer cell lines. Process diagram:
Proper citation: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Rizwan Ansari, Fiona Behan, Mathew Garnett 2020. Blasticidin titration of cancer cell lines. protocols.io dx.doi.org/10.17504/protocols.io.bgz6jx9e Copy
Authors: Benjamin Bolduc
Group: Sullivan Lab
Summary: A collection of protocols designed to guide the user in processing a viral metagenome from raw sequence data to assembly, and subsequent analysis. The user uses actual reads from Ocean Sampling Day (2014) and processes them entirely within Cyverse, a NSF-supported cyberinfrastructure.
Proper citation: Benjamin Bolduc 2016. Applying vContact to Viral Sequences and Visualizing the Output (Cyverse). protocols.io dx.doi.org/10.17504/protocols.io.ev8be9w Copy
Authors: Renata Alitto, Michela Borges, Letícia Dias de Oliveira, Karin Regina Seger
Summary: DNA extraction, amplification, and sequencing, particularly for the brittel star Ophiothrix.
Proper citation: Renata Alitto, Michela Borges, Letícia Dias de Oliveira, Karin Regina Seger 2019. DNA extraction, amplification, and sequencing of Ophiothrix (Echinodermata: Ophiuroidea). protocols.io dx.doi.org/10.17504/protocols.io.tz7ep9n Copy
Authors: Kenneth M. Stedman, Kate Porter, and Mike L. Dyall-Smith
Group: VERVE Net
Summary: This is a protocol from: Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. 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.
Proper citation: Kenneth M. Stedman, Kate Porter, and Mike L. Dyall-Smith 2016. Isolation of Haloviruses from Natural Waters. protocols.io dx.doi.org/10.17504/protocols.io.ebcbaiw Copy
Authors: Josh Quick
Group: Diaz-Munoz Lab, Coronavirus Method Development Community, Szentagothai Research Centre Virology
Summary: This is a subprotocol for performing adapter ligation with AMII
Proper citation: Josh Quick 2020. Adapter ligation with AMII. protocols.io dx.doi.org/10.17504/protocols.io.bdp9i5r6 Copy
Authors: Shripal Gandhi, Santo Purnama, Keyur Patel, Praveen Sukumara, Dr Rinu R Ravi
Group: Coronavirus Method Development Community, SensingSelfMission, XPRIZE Rapid Covid Testing
Summary: Summary:The S1 COVID-19 Rapid Antigen test kit developed by Sensing Self Pte. Ltd. can quickly and qualitatively detect the spike glycoprotein of novel coronavirus (SARS-COV-2) in human saliva/sputum/stool samples. It can be used as an aid for COVID-19 diagnosis.Objective:According to the clinical trial plan, the S1 COVID-19 Rapid Antigen test kit or “test reagent”, is to test saliva/sputum/stool samples from healthy subjects and confirmed COVID-19 patients. Test results are compared with another commercial SARS-COV-2 nucleic acid detection kit with CFDA approval, which is defined as the “gold standard”. The sensitivity, specificity, and total agreement rate are used to evaluate the feasibility of the test reagent in clinical applications.Method:A collection of clinical samples were examined by the S1 COVID-19 Rapid Antigen test kit and the gold standard SARS-COV-2 nucleic acid detection kit in parallel, to calculate the clinical sensitivity, clinical specificity and total agreement rate of the test reagent.Results:Compared to the gold standard, the clinical sensitivity of test reagent reached 90.0%, the clinical specificity reached 100.0%, and the total coincidence rate reached 95.0%.Conclusions:The performance of test reagent has a high agreement rate with the gold standard, proving its good feasibility in diagnosing suspected COVID-19 cases.
Proper citation: Shripal Gandhi, Santo Purnama, Keyur Patel, Praveen Sukumara, Dr Rinu R Ravi 2020. SensingSelf S1 Rapid Antigen Test (Saliva/Sputum/Stool). protocols.io dx.doi.org/10.17504/protocols.io.bj6ykrfw Copy
Authors: Ana Paula Santos, Thiago Mafort, Rogério Rufino
Summary: Thoracentesis is a procedure in which a needle is inserted into the pleural space between the lungs and the chest wall. This procedure is done to remove excess fluid from the pleural space or to determine the cause of the pleural effusion. The aim of this protocol is to spread to the cientific community the standard procedures to perform thoracentesis and pleural biopsy at HUPE / UERJ.
Proper citation: Ana Paula Santos, Thiago Mafort, Rogério Rufino 2018. Standardized procedures for thoracentesis / pleural biopsy at Pleural Diseases Outpatient Clinic - HUPE / UERJ. protocols.io dx.doi.org/10.17504/protocols.io.saceaaw Copy
Authors: Frederic Rimet, Marine Vautier, Rainer Kurmayer, Nico Salmaso, Camilla Capelli, Agnès Bouchez, Peter Hufnagl, Isabelle Domaizon
Group: EcoALpsWater
Summary: The objective of this protocol is to provide a reliable and replicable method for the sampling of river micro-phytobenthos and associated microbes in biofilms, to be used in both downstream DNA analysis and algal microscopic counts.
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The filed protocol is optimized for routine sampling and is in agreement with CEN guidance (NF EN 13946) and CEN technical report (CEN/TR 17245) for the analysis of benthic diatoms from rivers and lakes.
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The application proposed here in the context of EcoAlpsWater aims in comparing DNA inventories to traditional inventories (microscopy).
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This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium (involving 12 partners : http://www.alpine-space.eu/projects/eco-alpswater/en/home) have contributed to the optimization of this protocol.
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Phytobenthos-based ecological assessment of running waters is part of the EU Water Framework Directive 2000/60/EC (European Commission) allowing to evaluate the level of nutrients and organic matter pollution in rivers. According to the countries, phytobenthos assessment includes all groups of algae or is based only on diatoms communities. Other periphytic organisms such as fungi, bacteria or microbial eukaryotes as cilates or planktonic (metaphytic) algae are not considered in this assessment method. Further the group of charophytes is covered by the macrophyte ecology method assessment.
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The recent development of DNA metabarcoding has the potential to complement the traditional biological monitoring based on the direct observation of the organisms, both by reducing sample-processing cost and time and by offering the opportunity to consider a larger taxonomic diversity (e.g. bacteria, heterotrophic micro-eukaryotes …).
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The protocol described here is dedicated to the sampling of biofilms in rivers both for microscopic enumeration of the phytobenthic community and for downstream DNA analyses of microbial assemblages.
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This field protocol is based on routine methods used for biofilms sampling and is in agreement with:
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- CEN, 2014. Water quality - NF EN 13946 - Guidance for the routine sampling and preparation of benthic diatoms from rivers and lakes. Afnor, 1-23.
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- CEN 2018. Water quality - CEN/TR 17245 - Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses. CEN, 1-8.
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Proper citation: Frederic Rimet, Marine Vautier, Rainer Kurmayer, Nico Salmaso, Camilla Capelli, Agnès Bouchez, Peter Hufnagl, Isabelle Domaizon 2020. River biofilms sampling for both downstream DNA analysis and microscopic counts. protocols.io dx.doi.org/10.17504/protocols.io.ben6jdhe Copy
Authors: Yiheng Hu, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms, Team Schwessinger
Summary: This is a protocol optimized from Oxford Nanopore Technology 1D native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) for sequencing amplicons of fungal marker genes (ITS, EF1a) using PCR.Thanks to Dr. Benjamin Schwesinger for his suggestions about the protocol.Thanks to Dr. Leon Smith (ANU, Linde lab), Prof. Wieland Meyer (USydney) and Dr. Laszlo Irinyi for their contribution of fungal samples.
Proper citation: Yiheng Hu, Benjamin Schwessinger 2018. Amplicon sequencing using MinION optimized from 1D native barcoding genomic DNA. protocols.io dx.doi.org/10.17504/protocols.io.mhkc34w Copy
Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Proper citation: Angel Justiz-Vaillant 2020. An easy chromatographic method for purification of Immunoglobulin Y (IgY) using HiTrap™ Columns.. protocols.io dx.doi.org/10.17504/protocols.io.bju7knzn Copy
Authors: Tim Regan
Summary: This protocol describes the preparation of biological samples (specifically from a marine environment e.g. hatchery or RAS unit) for amplicon sequencing. Starting with a biological sample stored in Qiagen buffer ATL, or similar, it begins with a bead beating process to homogenise the sample. Enzymatic lysis using Metapolyzyme and Proteinase K are emplyed to ensure efficient DNA release. The Qiagen DNeasy kit is used to column extract DNA from lysates. Following concentration estimates of DNA elutions, samples are diluted >1:10 to avoid PCR inhibition during amplicon library preparation.
Proper citation: Tim Regan 2020. Preparing biological samples for metabarcoding. protocols.io dx.doi.org/10.17504/protocols.io.bms8k6hw Copy
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