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Authors: Maria Lucia Silva, Talita Gagliardi, Eurico Arruda
Summary: IF Protocol used to detect RSV in nasopharyngeal samples.
Proper citation: Maria Lucia Silva, Talita Gagliardi, Eurico Arruda 2019. Immunofluorescence Assay to detect respiratory syncytial virus. protocols.io dx.doi.org/10.17504/protocols.io.w8ufhww Copy
Authors: Matus Valach
Summary: A brief protocol for the preparation of glyoxal used in glyoxal/borate and RNase T1-based detection of inosine residues in RNAs.
Proper citation: Matus Valach 2016. Preparation of deionized glyoxal. protocols.io dx.doi.org/10.17504/protocols.io.ejsbcne Copy
Authors: Fatma Gomaa, Paulo Garcia, Jennifer Delany, Cullen Buie, Peter Girguis, and Virginia Edgcomb
Proper citation: Fatma Gomaa, Paulo Garcia, Jennifer Delany, Cullen Buie, Peter Girguis, and Virginia Edgcomb 2017. Transient genetic transformation of Bodo caudatus using square wave electroporation system and pUB-GFP plasmid. protocols.io dx.doi.org/10.17504/protocols.io.g5yby7w Copy
Authors: Lenny Teytelman
Summary: Transformation of heat-shock competent E. coli cells
Proper citation: Lenny Teytelman 2014. Transformation of competent E.coli cells with plasmid DNA. protocols.io dx.doi.org/10.17504/protocols.io.ccfstm Copy
Authors: Katie Pitz, Nathan Truelove, Charles Nye, Reiko P Michisaki, Francisco Chavez
Group: MBON eDNA
Summary: The 12S protocol is aimed at amplifying the hypervariable region of the mitochondrial DNA 12S rRNA gene in eukaryotes. The primers (MiFish-U-F & MiFish-U-R) used in this protocol were developed by Miya et al., 2015 for metabarcoding environmental DNA (eDNA) from fishes. Touchdown thermocycling protocols were adapted from the CALeDNA group.This work was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASA), and the U.S. Integrated Ocean Observing System (IOOS) Program Office.Citations1) Miya M et al. 2015 MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. R.Soc.opensci. 2: 150088. http://dx.doi.org/10.1098/rsos.1500882) CALeDNA. 2019. University of California Conservation Genomics Consortium.
Proper citation: Katie Pitz, Nathan Truelove, Charles Nye, Reiko P Michisaki, Francisco Chavez 2020. Environmental DNA (eDNA) 12S Metabarcoding Illumina MiSeq NGS PCR Protocol (Touchdown). protocols.io dx.doi.org/10.17504/protocols.io.bcppivmn Copy
Authors: Ian Simpson
Group: Diabetic Complications Consortium
Summary: This document describes the procedure for inducing a stroke in diabetic RCS10 mice, their non-diabetic controls and NON/ShiLtJ control mice. Our initial objective was to use those RCS10 mice that did not become diabetic as the controls for the experiment to compare the effect of diabetes on stroke outcome and that data is illustrated in Figure 2. However, with further breading the numbers of non- diabetic RCS 10 mice at twenty weeks of age were so few that it became necessary to use the NON/ShiLtJ mice which are 85% genetically identical to the RCS10 mice and stroke outcome is illustrated in Figure 3.Diabetic Complications:
Proper citation: Ian Simpson 2019. Induce Stroke via Hypoxia-Ischemia. protocols.io dx.doi.org/10.17504/protocols.io.8e5htg6 Copy
Authors: Maya Brewer, Yuantee Zhu, Elizabeth Neumann, Danielle Gutierrez, Jeff Spraggins, Mark De Caestecker
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:To describe the procedure for multiple cycles of immunofluorescence on human kidney tissue embedded in carboxymethylcellulose.Expected Outcome:Kidney tissue sections that have been tagged with antibodies for imaging microscopy.
Proper citation: Maya Brewer, Yuantee Zhu, Elizabeth Neumann, Danielle Gutierrez, Jeff Spraggins, Mark De Caestecker 2019. Multiplex Immunofluorescence on Fresh Frozen Tissue. protocols.io dx.doi.org/10.17504/protocols.io.665hhg6 Copy
Authors: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda
Group: Coronavirus Method Development Community
Summary: This protocol is folked from "ARTIC amplicon sequencing protocol for MinION for nCoV-2019" by Josh Quick to adapt it to illumina sequencers. Because the PCR products are fragmented and ligated with adapters, this protocol is not restricted to 250 PE mode of MiSeq. While the library preparation uses QiaSeq FX by Qiagen and is basically straight forward (as par kit instruction but set to 1/4 scale), some tweaks for much of simplicity and speed were added.Change from the previous version (V1): The amount of cDNA input to multiplex PCR is increased (now almost same amount to the ARTIC's original protocol) (Step 10). This change gives better results for samples with extremely low RNA copy. Amount of adapter solution input was corrected (Step 19).
Proper citation: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda 2020. nCoV-2019 sequencing protocol for illumina. protocols.io dx.doi.org/10.17504/protocols.io.betejeje Copy
Authors: Alise Ponsero, Bonnie Hurwitz
Group: Metafunc course 2018
Summary: This protocol explores how to run USEARCH (v11.0.667) (Edgar, 2010) on a 16s RNA dataset.USEARCH is a sequence analysis tool that offers search and clustering algorithms. Here USEARCH will be used to run a quality control on a 16S RNA dataset. This protocol uses a staggered mock community as an example (see attached files).
Proper citation: Alise Ponsero, Bonnie Hurwitz 2018. Run USEARCH for quality control of a 16s RNA dataset. protocols.io dx.doi.org/10.17504/protocols.io.s8iehue Copy
Authors: Integrated Islet Distribution Program
Group: Integrated Islet Distribution Program
Summary: This SOP defines the pancreas donor profile acceptable for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP). This SOP applies to all IIDP islet distribution centers using funds from the NIDDK to manufacture purified human pancreatic islets for basic research studies for IIDP approved investigators.The IIDP depends on the subcontracted human islet isolation centers to provide research investigators with human islets. It is the responsiblity of the human islet centers to obtain research quality pancreata from the Organ Procurement Organizations (OPO). Some criteria are stricter than those used by transplant centers for organ transplant donors. This protocol provides the inclusion and exclusion criteria in order to determine the proper parameters for research donors.The donor criteria also reflects the required 2020 testing for COVID-19. Positive testing for COVID-19, HIV, Hepatitis C, and Hepatitis B are examples of some exclusion criteria.References (attached)OPTN Policies Effective as of April 3 2020 [Emergency COVID-19 Policy Actions]Organ Procurement and Transplantation Network, 2020 Apr.2013 PHS Guideline for Reducing Human Immunodeficiency Virus, Hepatitis B Virus, and Hepatitis C Virus Transmission through Organ Transplantation1 Behavioral Risk Factors for Recent HIV, HBV, or HCV Infection. Organ Procurement and Transplantation Network, 2013 Nov.
Proper citation: Integrated Islet Distribution Program 2020. Determining IIDP Minimal Donor Criteria. protocols.io dx.doi.org/10.17504/protocols.io.bfrpjm5n Copy
Authors: Ken Christensen
Summary: Product DescriptionThe Zymoclean™ Gel DNA Recovery Kit provides a hassle-free method for high yield recovery of pure DNA from agarose gels. Simply add the specially formulated Agarose Dissolving Buffer (ADB) to the gel slice containing your DNA sample, let dissolve, and then transfer to the supplied Zymo-Spin™ Column. There is no need for organic denaturants or chloroform. Instead, the product utilizes Fast-Spin column technology to yield high-quality DNA in just 15 minutes. DNApurified using the Zymoclean™ Gel DNA Recovery Kit is perfectly suited for use in DNA ligation reactions, sequencing, DNA labeling reactions, PCR, etc.
Proper citation: Ken Christensen 2020. Zymoclean Gel DNA Recovery--CHEM 584. protocols.io dx.doi.org/10.17504/protocols.io.bmyzk7x6 Copy
Authors: Moriah Beck
Group: Beck Lab
Summary: Second-derivative spectroscopy has proven to be an effective analytical tool because of its ability to resolve overlapping bands in the normal spectrum.This protocol is used to determine the degree of tyrosine exposure in proteins using second-derivative UV spectroscopy.
Proper citation: Moriah Beck 2019. Second-derivative UV Spectroscopy. protocols.io dx.doi.org/10.17504/protocols.io.q7qdzmw Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is a protocol for a typical DNase I Reaction, using the M0303 RNase-free DNase I.
Proper citation: New England Biolabs 2015. A Typical DNase I Reaction (M0303). protocols.io dx.doi.org/10.17504/protocols.io.cqevtd 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.9vgh63w Copy
Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol was designed and developed at this laboratory.The assay targets the capsid peptide coding region of DENV 1-4 and is desigend as a qualitative screening test for human cases of DENV infection.
Proper citation: Ian Mackay, Judy Northill 2018. Dengue virus (DENV) universal MGB TaqMan 2017. protocols.io dx.doi.org/10.17504/protocols.io.n7jdhkn Copy
Authors: Simon Blanchoud
Group: Blanchoud lab, UNIFR
Summary: Three alternative solutions for artificial seawater (ASW) have been tested successfully on our Botrylloides colonies. For routine work, we use commercial sea salts (CSS), for most analyses we use the Cold Spring Harbor Protocols (CSPH) and for very clean work the K-depleted Phosphate-buffered saline (K-PBS). While, CSPH and K-PBS can be prepared at 10X, CSS should be prepared at 2X directly.Mix to dissolve and adjust pH as well as salinity to your local conditions.
Proper citation: Simon Blanchoud 2021. Artificial sea water . protocols.io dx.doi.org/10.17504/protocols.io.brxbm7in Copy
Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Proper citation: Matthew Sullivan 2016. g23 T4-type (myovirus) PCR Protocol. protocols.io dx.doi.org/10.17504/protocols.io.dfq3mv Copy
Authors: Benjamin Bolduc
Group: Sullivan Lab, iVirus
Summary: Preparing data for use in vContact by using VirSorted Ocean Sampling Day (2014) contigs, using tools available in Cyverse. This protocol creates a BLAST DB, BLASTs sequences, and creates a gene-to-contig mapping file. Results from this protocol are suitable for vContact-PCs.
Proper citation: Benjamin Bolduc 2017. Preparing Data for vContact from Proteins (Cyverse). protocols.io dx.doi.org/10.17504/protocols.io.gwdbxa6 Copy
Authors: Honghao Zhao, Jasmine Chong, Rong Tang, Li Li, Jianguo Xia, Dapeng Li
Group: Metabolomics Protocols & Workflows
Summary: The different kinds of stained histological sections are widely used to detect and observe physiological and pathological changes in various tissues.
Proper citation: Honghao Zhao, Jasmine Chong, Rong Tang, Li Li, Jianguo Xia, Dapeng Li 2018. Histological Observation of Tissues. protocols.io dx.doi.org/10.17504/protocols.io.smfec3n Copy
Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: A real-time RT-PCR to designed to detect SARS-CoV-2 and other related sarbecoviruses. Based on sequence MN908947 made available by Professor Yong-Zhen Zhang, Fudan University, Shanghai, China. The target region encodes the nucleocapsid (N).Tested on wild-type SARS-CoV-2 virus , it is expected to be capable of detecting SARS-CoV-2, bat-like SARS and SARS virus (members of the subgenus Sarbecovirus).Limit of detection not yet determined.The performance of the assay has not been tested with low viral load samples or samples from patients who are clinically well.The sensitivity of this assay was improved with the use of the SensiFast™Probe Lo-ROX One-step kit. A single 1 mismatch at probe-binding site identified with the BetaCoV/USA/CA1/2020|EPI_ISL_406034 (GenBank MN994467.1) variant of SARS-CoV-2 (as of 23JUNE2020).Probe is in the 3'-5' (reverse complement) direction.Reverse primers were replaced in March 2020.We also recommend the ORF1ab assay (Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR ORF1ab 2020), US-CDC-N1 assay or the E gene assay by Corman et al. (Protocol v2-1)Notes: Assay is optimised.This test has identified clinical positive cases of coronavirus disease (COVID-19)Notes: Assay is optimised.This test has identified clinical positive cases of coronavirus disease (COVID-19)
Proper citation: Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR N gene 2020 . protocols.io dx.doi.org/10.17504/protocols.io.bhpwj5pe Copy
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