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Authors: Lin Zhong, Jeffrey Rottman, Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The most common cause of cardiovascular mortality in man is the outcome from myocardial ischemic injury. Accordingly, it is necesarry to study the corresponding process of heart injury recovery in many mouse models relevant to human cardiovascular disease. This protocol describes the surgical induction of myocardial injury via transient occlusion of a coronary artery followed by reperfusion (ischemia-reperfusion injury).
Proper citation: Lin Zhong, Jeffrey Rottman, Chee Lim 2019. Vandy - Myocardial Ischemia Reperfusion. protocols.io dx.doi.org/10.17504/protocols.io.6xfhfjn Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to prepare Tris Buffered Saline (TNT). 10X TNT is the stock solution used to prepare 1X TNT, which equilibrates tissue and maintains pH in physiological range.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 2021. Tris Buffered Saline (TNT). protocols.io dx.doi.org/10.17504/protocols.io.brdkm24w Copy
Authors: Alexandra Ley
Summary: Total genomic DNA was extracted from leaf tissue to amplify and sequence individually five specific target loci from nuclear and chloroplast genome.
Proper citation: Alexandra Ley 2018. DNA amplification. protocols.io dx.doi.org/10.17504/protocols.io.taneide Copy
Authors: Benjamin Daniel
Summary: A protocoll outlining the Preperation and transformation of electro-competent cells for Vibrio natriegensPreparation of electrocompetent cells (Vibrio natrigens)10 mL BHI (Brain haert infusion) + v2 salts Overnight cultureInoculation of a new BHI + v2 media with 1% of the overnight culture as inoculum.Grow at 37°C shaking to an OD of 0.5Chill the culture on ice for 15 minUse a chilled (4°C) centrifuge at 4,500 r.p.m. for 20 minDecant the supernatant diligently and carefullyResuspend gently in 5-10 mL Electroporation-Buffer (680 mM sucrose, 7 mM K2HPO4, pH 7), then fill the falcon tube to the top.To wash, spin again at 4.500 r.p.m. for 15 min at 4 °C, then resuspend gently in 5 mL Electroporation-Buffer. Repeat washing twice for a total of three times.Spin again at 4.500 r.p.m. for 15 min at 4 °C, decant supernatant and resuspend carefully in the residual buffer.Adjust volume for a OD of 16Aliquot in chilled Micro-Reaction-Tubes, then snap freeze in liquid nitrogenStore at –80°C
Electroporation remove one aliquot with electrocompetent cells from storagekeep on ice until thawedadd plasmid and mix gentlyTransfer to chilled electroporation cuvetteElectroporation is done with the following parameters: 700(–900) V, 25 μF, 200 Ω in a 1mm cuvetteRecover for one hour at 37°C in brain heart infusion with v2 saltsPlate on LB2 agar plates.
Electroporationremove one aliquot with electrocompetent cells from storagekeep on ice until thawedadd plasmid and mix gentlyTransfer to chilled electroporation cuvetteElectroporation is done with the following parameters: 700(–900) V, 25 μF, 200 Ω in a 1mm cuvetteRecover for one hour at 37°C in brain heart infusion with v2 saltsPlate on LB2 agar plates.
Proper citation: Benjamin Daniel 2018. Electroporation of Vibrio natriegens (Weinstock et al. 2016, modified .). protocols.io dx.doi.org/10.17504/protocols.io.ptmdnk6 Copy
Authors: Camille Poirier, Raquel Rodríguez‐Martínez, Emily Cook, David Milner, Alexandra Z. Worden, Thomas A. Richards
Group: Worden Lab, Richards Lab
Proper citation: Camille Poirier, Raquel Rodríguez‐Martínez, Emily Cook, David Milner, Alexandra Z. Worden, Thomas A. Richards 2019. Cell sorting of marine heterotrophic flagellates for single-cell genome amplification. protocols.io dx.doi.org/10.17504/protocols.io.ywpfxdn Copy
Authors: Jana Ordon, Hannelore Espenhahn, Carola Kretschmer, Johannes Stuttmann
Summary: Detailed protocol with pictures for the transformation of Nicotiana benthamiana. The same protocol will also work for transformation of Nicotiana tabaccum, but regeneration of transformed plants will take more time.
Proper citation: Jana Ordon, Hannelore Espenhahn, Carola Kretschmer, Johannes Stuttmann 2018. Stable transformation of Nicotiana benthamiana. protocols.io dx.doi.org/10.17504/protocols.io.sbaeaie Copy
Authors: Adrien Assie
Group: protocols.io news
Summary: Here is a short explanation on how to add a work in progress protocol to a private group. This protocol shows how to add a protocol editable by multiple people to edit it before its final publication on protocols.ioThis is also an example protocol on how to explain to the members of the group on how to share protocols.
Proper citation: Adrien Assie 2018. Share a WIP protocol with a private group. protocols.io dx.doi.org/10.17504/protocols.io.qzhdx36 Copy
Authors: Rabia Khan
Proper citation: Rabia Khan 2016. Splenocyte Preperation. protocols.io dx.doi.org/10.17504/protocols.io.es9beh6 Copy
Authors: Christina Alba, Melissa Islam
Group: Denver Botanic Gardens Research and Conservation
Summary: Floristic surveys and plant collections represent baseline data critical to understanding plant species diversity and distributions. To make plant collections valuable for current and future researchers, both the physical specimen and all associated data must be of the highest quality. This protocol describes the steps by which researchers at Denver Botanic Gardens make vascular plant specimen collections and record associated data.
Proper citation: Christina Alba, Melissa Islam 2019. Field Collecting Protocol for Vascular Plants. Denver Botanic Gardens. Kathryn Kalmbach Herbarium of Vascular Plants. protocols.io dx.doi.org/10.17504/protocols.io.4f4gtqw Copy
Authors: Sara Beier, Sara Beier
Summary: A prerequisite to improve the predictability of microbial community dynamics is to understand their assembly mechanisms. To study factors that contribute to microbial community assembly, we examined temporal dynamics of genes in five aquatic metagenome time series, originating from marine off-shore or coastal sites and one lake, while focusing on a trait-based data evaluation.We expected to find gene-specific patterns for the temporal allele variability depending on the metacommunity size of carrier-taxa and variability of the milieu and the substrates that the resulting enzymes are exposed to. In more detail we hypothesized that a larger metacommunity would cause increased temporal variability of functional units, as shown previously for taxonomic units. Furthermore, we hypothesized that multi-copy genes feature higher temporal variability then single-copy genes, because gene multiplication is often the consequence of increased variability in (subtil) changes of substrate quality and quantity. Finally, we hypothesized that direct exposure of proteins to the extracellular environment would result in increased temporal variability of the respective gene compared to intracellular proteins as they would be exposed to highly variable conditions. The first two hypotheses were confirmed in all, while an effect of the subcellular location of gene-products was only seen in three out of the five time series. The gene with highest allele variability throughout all datasets was an iron transporter, which also represents a target for phage infections. This finding points to the general importance of iron transporter mediated phage infections on the assembly and maintenance of diversity of aquatic prokaryotes.
Proper citation: Sara Beier, Sara Beier 2020. allele.variability. protocols.io dx.doi.org/10.17504/protocols.io.bkhqkt5w Copy
Authors: Denville Scientific
Group: Denville Scientific, Inc.
Summary: Denville LE Agarose is an all purpose agarose for routine nucleic acid electrophoresis of fragments between 500bp-23,000 bp.Denville LE Agarose has no detectable DNase or RNase activity.
Proper citation: Denville Scientific 2016. Microwave Instructions for Agarose Preparation (for Denville® Agarose LE). protocols.io dx.doi.org/10.17504/protocols.io.gsbbwan Copy
Authors: Andreea S
Group: iGEM Groningen 2020
Summary: Introducing NLP14a in the genome of B. mycoides: In order to create plasmid pYCR-gamyNLP, the backbone of Pycr will be digested with the PCR product of gamy_Fw and gamy_Rv to produce the pYCR_gamy (CRISPR vector containing the gRNA). The NLP14a sequence will be ordered from Twist Biosciences And PCR-amplified with the primers NLP_Fw and NLP_Rv. The resulting PCR product, as well as the pYCR_gamy vector will be digested with SfiI. The digested mixtures will be ligated using T4 ligase. Kill switch cloning: The suggested kill switch mechanism is based on Trp auxotrophic strains as well as a toxin-antitoxin mechanism. The toxin will be introduced in the genome using crispr in place of trpE. The antitoxin will be maintained as a cytoplasmic plasmid (pAD-YqcF) and expressed only in the presence of solanine. Unfortunately, to the best of our knowledge, no operator that binds solanine of B. mycoides has been described in literature. If it is provebn that no such operator is present, we suggest on oly keeping the tryptophan dependence strategy. The plasmid pAD-KPS12-Pman will used as a starting point for introducing the antitoxin gene (ygcF) in B. mycoides. The vector will be cut with XbaI and SphI and ligated with the PCR amplified ygcF (using ygcF_Fw and ygcF_Rv as primers). In order to make the antitoxin expression inducible by solanine we plan on replacing the mannose inducible promoter with a promoter induced by solanine. To accomplish this, we will use the primers Psol_Fw and Psol_Rv to amplify the solanine promoter. The pAD-ygcFvector will be digested with EcoRI and XbaI, and ligated with the solanine promoter PCR product that will be digested as well with the up mentioned restriction enzymes. In order to create plasmid pYCR-gtrpEYqcG plasmid, the backbone of Pycr will be digested with BsaI and ligated with the annealing product of gtrpe_Fw and gtrpe_Rv to produce the pYCR_gtrp (CRISPR vector containing the gRNA). Synthetic dna for yqcG will be ordered from Twist Biosciences and PCR-amplified with the primers Yqcg_Fw and Ygcg_Rv. The resulting PCR product, as well as the pYCR_gtrpE vector will be digested with SfiI. The digested mixtures will be ligated using T4 ligase.
Proper citation: Andreea S 2020. Cloning of Bacillus mycoides . protocols.io dx.doi.org/10.17504/protocols.io.bkuwkwxe Copy
Authors: Sebastian Bassi
Summary: This protocol tells how to fix the computer
Proper citation: Sebastian Bassi 2017. Fix the computer. protocols.io dx.doi.org/10.17504/protocols.io.ka8cshw Copy
Authors: Benjamin Tully
Group: Center for Dark Energy Biosphere Investigations
Summary: The workflow applied to Tara Oceans raw sequence data for generating assemblies suitable for genomic binning.Used in:"290 Metagenome-assembled Genomes from the Mediterranean Sea: Ongoing Effort to Generate Genomes from the Tara Oceans Dataset" - bioRxiv https://doi.org/10.1101/069484"Undocumented potential for primary productivity in a globally-distributed bacterial photoautotroph" - submitted
Proper citation: Benjamin Tully 2017. Assembly Procedure Applied to TARA Oceans Data (Ex. North Pacific). protocols.io dx.doi.org/10.17504/protocols.io.hfqb3mw Copy
Authors: P. Dreux Chappell, Bethany D. Jenkins
Summary: A protocol to separate chloroplasts from diatom cells using ammonium fluoride to permeate the silica frustrule and a percoll gradient to separate the plastid from other cellular components.
Proper citation: P. Dreux Chappell, Bethany D. Jenkins 2016. Diatom Chloroplast Isolation Steps. protocols.io dx.doi.org/10.17504/protocols.io.ea3bagn Copy
Authors: Angel Justiz-Vaillant, Belkis Ferrer-Cosme
Group: University of the West Indies, [email protected]
Summary: Granulocyte macrophage-colony stimulating factor (GM-CSF) is a monomeric glycoprotein. It is a cytokine secreted by macrophages, T cells, natural killer cells, mast cells, endothelial cells and fibroblasts. It acts as a growth factor. [1] Reference1. Egea L, Hirata Y, Kagnoff MF. GM-CSF: a role in immune and inflammatory reactions in the intestine. Expert Rev Gastroenterol Hepatol. 2010 Dec;4(6):723-31. doi: 10.1586/egh.10.73. PMID: 21108592; PMCID: PMC3291482.
Proper citation: Angel Justiz-Vaillant, Belkis Ferrer-Cosme 2020. ELISA for quantification of granulocyte macrophage-colony stimulating factor (GM-CSF) in tissue culture supernatant, human serum or plasma.. protocols.io dx.doi.org/10.17504/protocols.io.bktykwpw Copy
Authors: Code Ocean, Addgene, protocols.io
Group: Tools and Resources for Reproducibility
Summary: This is a handout that was created by Code Ocean, Addgene, and protocols.io for the Caltech workshop on reproducibility. For more information and slides from the workshop, please see: https://codeocean.com/workshop/caltech.Please feel free to clone and modify it. If you do, would be wonderful to see you share the new resource in this group. Also, please suggest other useful resources.
Proper citation: Code Ocean, Addgene, protocols.io 2018. Caltech Workshop Reproducibility Handout (from CodeOcean, Addgene, protocols.io). protocols.io dx.doi.org/10.17504/protocols.io.nibdcan Copy
Authors: Brian Baltzar
Summary: Estimate a protein concentration in a solution using the Bradford protein assay
Proper citation: Brian Baltzar 2017. Bradford Protein Concentration determination. protocols.io dx.doi.org/10.17504/protocols.io.girbud6 Copy
Authors: 宏亮 董
Group: 2019 iGEM NEFU_China
Proper citation: 宏亮 董 2019. PCR. protocols.io dx.doi.org/10.17504/protocols.io.7hyhj7w Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the correct protocol if you are using the C2987I cells. If you are using the C2987H cells, please refer to this protocol.
Proper citation: New England Biolabs 2014. High Efficiency Transformation Protocol (C2987I). protocols.io dx.doi.org/10.17504/protocols.io.chht35 Copy
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