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Authors: Ana Cristina Jaramillo Madrid, Justin Ashworth
Summary: This protocol has been successfully used to express nourseothricin resistance gene, mVenus fluorescence protein and other proteins related to triterpenoids production in Thalassiosira pseudonana (Tp) strain CCMP1335. The original protocol was published by Karas et al. (2015) where a detailed description of L1 medium and plates preparation is presented.
Proper citation: Ana Cristina Jaramillo Madrid, Justin Ashworth 2019. Tranformation of Thalassiosira pseudonana via bacterial conjugation. protocols.io dx.doi.org/10.17504/protocols.io.7fghjjw Copy
Authors: Pradeep Rajendran, Guy Salama, Ching Zhu, Peter Hanna
Group: SPARC
Proper citation: Pradeep Rajendran, Guy Salama, Ching Zhu, Peter Hanna 2020. Dual Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart during Optogenetic Stimulation of the ICNS. protocols.io dx.doi.org/10.17504/protocols.io.bcdtis6n Copy
Authors: Patrick Tso
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
Oxygen consumption and carbon dioxide production is measured using indirect calorimetry. The Columbus Instruments Oxymax Equal Flow System is an indirect open circuit calorimeter designed to simultaneously measure metabolic performance of multiple subjects that have similar ventilation needs. uses an open circuit calorimetry technique. This system allows sixteen animal cages to be simultaneously monitored. Variables provided by this measurement include VO2, VCO2, RQ, and HEAT.
Proper citation: Patrick Tso 2019. U Cinn - Energy Expenditure Measurements. protocols.io dx.doi.org/10.17504/protocols.io.xi9fkh6 Copy
Authors: Benjamin Schwessinger and Megan McDonald
Group: Solomon Lab - Australian National University, High molecular weight DNA extraction from all kingdoms, Zymoseptoria community protocols (STBnet)
Summary: Extraction of high quality DNA for long read sequencing e.g. PacBio and MinIonOptimized for DNA extraction from Bipolaris sorokiniana. Also tested on Parastaognospora nodorum, Zymoseptoria tritici, wheat stripe rust, barley stripe rust and Pyrenophora tritici-repentisBuffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.Critical steps to obtain high quality DNA:Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.Do NOT incubate samples with KAc for prolonged time periodsPerform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction.
Proper citation: Benjamin Schwessinger and Megan McDonald 2017. High quality DNA from Fungi for long read sequencing e.g. PacBio, Nanopore MinION. protocols.io dx.doi.org/10.17504/protocols.io.k6qczdw Copy
Authors: David Eccles
Summary: This protocol is for a semi-manual method for read demultiplexing, as used after my presentation Sequencing DNA with Linux Cores and Nanopores to work out the number of reads captured by different barcodes.Input: reads as a FASTQ file, barcode sequences as a FASTA fileOutput: reads split into single FASTQ files per target [barcode]Note: barcode / adapter sequences are not trimmed by this protocol
Proper citation: David Eccles 2019. Demultiplexing Nanopore reads with LAST. protocols.io dx.doi.org/10.17504/protocols.io.42dgya6 Copy
Authors: Debora Ferreira Barreto-Vieira, Fernanda Cunha Jácome, Marcos Alexandre Nunes da Silva, Gabriela Cardoso Caldas, Elen Mello de Souza, Audrien Alves Andrade, Ortrud Monika Barth.
Proper citation: Debora Ferreira Barreto-Vieira, Fernanda Cunha Jácome, Marcos Alexandre Nunes da Silva, Gabriela Cardoso Caldas, Elen Mello de Souza, Audrien Alves Andrade, Ortrud Monika Barth. 2017. Cell fixation for processing for analysis by transmission electron microscopy. protocols.io dx.doi.org/10.17504/protocols.io.jqucmww Copy
Authors: Michael Van Dyke, James Cox
Summary: Short DNA probes, for example, those used in characterizing protein-DNA complexes, have historically been radiolabeled with 32P to allow their detection and quantitation following native polyacrylamide gel electrophoresis (PAGE). For reasons of economy or safety, alternative means of DNA detection need to be used. Described here is the resolution of 5' IRD700 and IRD800 end-labeled DNAs by native PAGE and their visualization and quantitation by IR fluorescence imaging.
Proper citation: Michael Van Dyke, James Cox 2017. DNA Analysis by Native Polyacrylamide Gel Electrophoresis and Infrared Fluorescence Imaging. protocols.io dx.doi.org/10.17504/protocols.io.mcyc2xw Copy
Authors: Hiraku Miyagi, Michio Hiroshima, Yasushi Sako
Summary: Immunofluorescence microscopy is used to determine molecular localization and to measure fluorescence intensity of activity in cultured cells. Reagents for fixation and/or permeabilization must be suitable for the molecule of interest. We developed a procedure to immunostain phosphatidylinositol biphosphate (PIP2) and phosphorylated Extracellular signal-Regulated Kinase (ERK) simultaneously. PIP2 is a phospholipid component of cell membranes. ERK is a mitogen-activated protein kinase (MAPK) that localized in the cytoplasm and nucleus after phosphorylation triggered by ligand activation. Different compounds are used to permeabilize cells in order to stain for these two molecules. Digitonin is used for PIP2 and Triton X-100 is used for phosphorylated ERK. However, since Triton X-100 can solubilize cell membrane lipids, it is not suitable for PIP2 labeling. In order to confer resistance against Triton X-100, we devised a method to fix anti-PIP2 antibody on the cell membrane antigen before permeabilization with Triton X-100. We modified a conventional immunostaining method to include secondary fixation and permeabilization after anti-PIP2 antibody labeling. The secondary fixation probably immobilizes antibodies bound to PIP2 on the cell membrane, perhaps by cross-linking them to surrounding protein molecules. Whatever the mechanism, this fixation enables PIP2-antibody complexes to remain on the membrane, despite treatment with Triton X-100, which is used to visualize phosphorylated ERK. Since PIP2 and ERK are major cell signaling molecules in various cellular phenomena, simultaneous detection and visualization of their phosphorylation states by this method should be useful for many studies.
Proper citation: Hiraku Miyagi, Michio Hiroshima, Yasushi Sako 2019. Double immunostaining for PIP2 and phosphorylated ERK. protocols.io dx.doi.org/10.17504/protocols.io.9gxh3xn Copy
Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Details of the conjugation episome used to generate the TpSil3p-EGFP expressing Thalassiosira pseudonana strain as reported in Faktorová et al (2019). The file contains:1/ a vector scheme summarizing all of its elements2/ expression cassette elements and their nucleotide sequences3/ fusion protein amino acid sequence4/ full vector DNA sequence with expression cassette elements annotated
Proper citation: Jernej Turnsek 2019. Faktorová_et_al_2019_pTpPuc3_TpSil3p-eGFP. protocols.io dx.doi.org/10.17504/protocols.io.8bdhsi6 Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes ddPCR titration of Lentivirus vectors. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/lentivirus-ddpcr-titration/.This protocol was modified from the publication Wang Y, Bergelson S, Feschenko M, 2018. Sample Data: When analyzing data there should be a clear distinction between negative droplets (black) and positive droplets (blue/green).The concentration of RRE positive droplets in the untransduced control should be close to zero (A01).In this protocol, the lentiviral particles are serially diluted and used to transduce HEK293T cells. Genomic DNA is extracted from the target cells and assayed for integrated copies of RRE. Since the samples that are assayed are diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across the dilutions. RPP30 copies should be relatively constant across samples.In the RRE example below, 2-fold serial dilutions of a sample were loaded in wells B01-H01.As shown in the image and table below, the concentration of RRE positive droplets increases by a factor of ~2 as you progress from the higher dilutions to the lower dilutions (blue).The concentration of RPP30 positive droplets stays relatively even across samples (green).To increase the accuracy of the titer, calculate and average of several dilutions.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. ddPCR Titration of Lentivirus Vectors. protocols.io dx.doi.org/10.17504/protocols.io.be7ijhke Copy
Authors: Lisa-Maria Rosenthal, Giang Tong, Katharina Schmitt
Summary: RNA concentration measurement and non-denaturing agarose gel electrophoresis
Proper citation: Lisa-Maria Rosenthal, Giang Tong, Katharina Schmitt 2019. RNA concentration measurement and non-denaturing agarose gel electrophoresis. protocols.io dx.doi.org/10.17504/protocols.io.2v5ge86 Copy
Authors: David Eccles
Summary: This protocol is for comparing different samples at the transcript level, using long reads that are mapped to transcripts.Input(s): demultiplexed fastq files (see protocol Demultiplexing Nanopore reads with LAST), transcript reference fasta file, annotation fileOutput(s): transcript table, sorted by differential coverage, annotated with gene name / description / location
Proper citation: David Eccles 2019. Stranded Transcript Count Table Generation from Long Reads. protocols.io dx.doi.org/10.17504/protocols.io.xu4fnyw Copy
Authors: Roscoff Culture Collection
Group: Roscoff Culture Collection
Summary: Medium used for some eukaryotesAdapted from CCAP web site : https://www.ccap.ac.uk/media/documents/BB.pdfSee also : http://www.ife.ac.uk/ccap/MediaRecipes.html#bb
Proper citation: Roscoff Culture Collection 2019. Bold SW 1 NV modified medium. protocols.io dx.doi.org/10.17504/protocols.io.s23eggn Copy
Authors: Justin Ryan, Nicole Coufal, Sage Aronson, Kelsey Ladt, Catelyn Andersen, Mark Zeller, Stephen Rawlings, Denise Malicki, Gene Yeo
Group: Coronavirus Method Development Community
Summary: The global pandemic due to SARS-CoV2 virus, also known as COVID-19, has drastically increased the need for nasopharyngeal-swab-based testing resulting in shortages of commercially available nasopharyngeal (NP) swabs. One solution to overcome the national deficit of swabs is for medical device manufacturers and hospitals to generate NP swabs. Numerous entities are attempting to manufacture a direct from 3D printing NP swab but presented here is a validated two-part swab manufacturing protocol utilizing 3D printing and manual intervention (wrapping of nylon fibers). We recommend material extrusion or powder bed fusion technologies utilizing materials that can be sterilized using high level heat decontamination. Through the application of 3D printing and manual fabrication, we present an NP swab that can be created in a controlled environment. Coupled with CDC published viral transport media, the following swab can be used for COVID-19 testing or for testing for other respiratory viruses (eg, influenza, respiratory syncytial virus). The CDC viral transport medium has only four reagents which are readily available.
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Proper citation: Justin Ryan, Nicole Coufal, Sage Aronson, Kelsey Ladt, Catelyn Andersen, Mark Zeller, Stephen Rawlings, Denise Malicki, Gene Yeo 2020. 3D Printed Nasopharyngeal Swabs with Wrapped Rayon Fibers Developed and validated by SCREEN (San Diego Covid19 Research Enterprise Network). protocols.io dx.doi.org/10.17504/protocols.io.bemxjc7n Copy
Authors: Wenlu Yang
Group: High molecular weight DNA extraction from all kingdoms
Proper citation: Wenlu Yang 2017. CTAB DNA Extraction Protocol of P. pruinosa. protocols.io dx.doi.org/10.17504/protocols.io.icgcatw Copy
Authors: Sarah Romac
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff
Summary: Radiolaria are protists which can't be cultivated. These microoganisms have to be isolated by single-cell for genetic identification and it can be difficult to get clean DNA.Here we optimized a DNA extraction protocol from protist single-cell.It works very well on single-cell Radiolaria, Foraminifers, but also Ciliates, Dinoflagelletes, Diatoms.
Proper citation: Sarah Romac 2020. Nucleic acids extraction from single cell using MasterPure Complete DNA purification (Epicenter). protocols.io dx.doi.org/10.17504/protocols.io.bekbjcsn Copy
Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol used to dissociate adult (8-10 wk) mouse spleen into single cells. Attained >95% viability, a variety of cell sizes, and ~10 million cells from 12 mg tissue.
Proper citation: Andrew Potter 2018. Adult Mouse Spleen Dissociation (On ice). protocols.io dx.doi.org/10.17504/protocols.io.q5cdy2w Copy
Authors: David Eccles
Summary: This protocol is for a semi-manual method for read demultiplexing, as used after my presentation Sequencing DNA with Linux Cores and Nanopores to work out the number of reads captured by different barcodes.Input: reads as a FASTQ file, barcode sequences as a FASTA fileOutput: reads split into single FASTQ files per target [barcode]Note: barcode / adapter sequences are not trimmed by this protocol
Proper citation: David Eccles 2019. Demultiplexing Nanopore reads with LAST. protocols.io dx.doi.org/10.17504/protocols.io.533g8qn Copy
Authors: ayten
Group: XPRIZE Rapid Covid Testing
Proper citation: ayten 2020. Easy Covid LAMP. protocols.io dx.doi.org/10.17504/protocols.io.bk6gkzbw Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The NEBNext Ultra II FS DNA Module contains the enzymes and buffers required to convert a broad range of input amounts of intact DNA into fragmented DNA with 5´ phosphorylated 3´ dA-tailed ends. The module is optimized for use with the NEBNext Ultra II Ligation Module (NEB #E7595) and with the NEBNext Ultra II Q5 Master Mix (NEB #M0544) if amplification is required. The fast, user-friendly workflow has minimal hands on time.
Proper citation: New England Biolabs 2020. E7805 NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® Protocol for use with Inputs ≤ 100 ng. protocols.io dx.doi.org/10.17504/protocols.io.k8tczwn Copy
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