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Authors: Tongbram Roshni Devi, Madhumita Dasgupta, MANAS RANJAN SAHOO, Paresh Chandra Kole, Narendra Prakash
Group: ICAR Research Complex for North Eastern Hill Region
Summary: A protocol for high-frequency direct organogenesis from root explants of Citrus jambhiri Lush. was developed. Full-length roots (~3cm) were isolated from the in vitro grown seedlings and cultured on Murashige and Skoog basal medium supplemented with Nitsch vitamin (MSN) with different concentrations of cytokinin [6-benzylaminopurine, (BAP)] and gibberellic acid (GA3). The frequency of multiple shoot proliferation was very high, with an average of 34.3 shoots per root explant when inoculated on the MSN medium supplemented with BAP (1.0 mg L–1) and GA3 (1.0 mg L–1). Optimal rooting was induced in the plantlets under half strength MSN medium supplemented with indole-3-acetic acid (IAA, 0.5-1.0 mg L–1). IAA induced better root structure than 1-naphthaleneacetic acid (NAA), which was evident from the result of scanning electron microscopy (SEM). The expressions of growth-regulating factor genes (GRF1 and GRF5) and GA3 signaling genes (GA2OX1 and KO1) were high in the regenerants obtained using MSN+BAP (1.0 mg/L)+GA3 (1.0 mg/L). The expressions of auxin regulating genes were high in roots obtained in ½ MSN+IAA1.0 mg L-1. Furthermore, the virus indexing of the regenerants confirmed that there were no virus amplicons detected for Huanglongbing and Citrus tristezavirus. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers detected no polymorphic bands amongst the regenerated plants. The high-frequency direct regeneration protocol in the present study provides an enormous significance in Citrus organogenesis, it’s commercial cultivation and genetic conservation.
Proper citation: Tongbram Roshni Devi, Madhumita Dasgupta, MANAS RANJAN SAHOO, Paresh Chandra Kole, Narendra Prakash 2021. Root-to-shoot organogenesis in Citrus jambhiri Lush.. protocols.io dx.doi.org/10.17504/protocols.io.bq4emyte Copy
Authors: BioLegend, Inc.
Group: BioLegend
Summary: This is a collection of BioLegend T Cell Activation with anti-CD3ε Antibodies protocols, for human and mouse.
Proper citation: BioLegend, Inc. 2016. T Cell Activation with anti-CD3? Antibodies. protocols.io dx.doi.org/10.17504/protocols.io.ezybf7w Copy
Authors: Jacob Beal, Traci Haddock-Angelli, Markus Gershater, Vishal Sanchania, Russell Buckley-Taylor, Geoff Baldwin, Natalie Farny, Richard Tennant, Paul Rutten
Group: iGEM Measurement
Summary: You will prepare a dilution series of monodisperse silica microspheres and measure the Abs600 in your plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement will allow you to construct a standard curve of particle concentration which can be used to convert 600 nm absorbance measurements into an estimated equivalent number of cells.
Proper citation: Jacob Beal, Traci Haddock-Angelli, Markus Gershater, Vishal Sanchania, Russell Buckley-Taylor, Geoff Baldwin, Natalie Farny, Richard Tennant, Paul Rutten 2019. Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles. protocols.io dx.doi.org/10.17504/protocols.io.549g8z6 Copy
Authors: Karen Cristine Gonçalves Dos Santos, Megan Mcdonald
Group: High molecular weight DNA extraction from all kingdoms
Summary: Adaptation of the protocol dx.doi.org/10.17504/protocols.io.k6qczdwOptimized for DNA extration from Bipolaris sorokiniana spores.
Proper citation: Karen Cristine Gonçalves Dos Santos, Megan Mcdonald 2018. High molecular weight DNA extraction for long read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.uppevmn Copy
Authors: Wenlu Yang
Proper citation: Wenlu Yang 2017. The draft genome sequence of a desert tree Populus pruinosa. protocols.io dx.doi.org/10.17504/protocols.io.ii5ccg6 Copy
Authors: Cristian Riccio
Summary: This protocol describes how to extract total RNA from C. elegans worms starting with 3300 worms on a 90mm NGM plate
Proper citation: Cristian Riccio 2019. Extracting total RNA from Caenorhabditis elegans using phase-lock gel separation tubes. protocols.io dx.doi.org/10.17504/protocols.io.5sug6ew Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used for the large-scale production of EnvA-pseudotyped recombinant rabies virus.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. Production of Recombinant EnvA Rabies Virus. protocols.io dx.doi.org/10.17504/protocols.io.bdvvi666 Copy
Authors: Lisa-Maria Rosenthal, Giang Tong, Katharina Schmitt
Proper citation: Lisa-Maria Rosenthal, Giang Tong, Katharina Schmitt 2019. Human RBM3 ELISA. protocols.io dx.doi.org/10.17504/protocols.io.3p5gmq6 Copy
Authors: Gergana Vandova
Group: Genomes to Natural Products Network
Summary: Gibson assembly is a simple, robust method for assembling multiple DNA fragments without restriction-ligation cloning. Our group routinely uses this method for assembling multiple fragments of DNA into larger constructs, in one step. We generally use Gibson assembly for assemblies of up to ~5 fragments and final construct size of ~20kb; for larger assemblies we usually use yeast homologous recombination.
The method Introduction from Daniel Gibson, et. al., is as follows:
'An isothermal, single-reaction protocol for assembling multiple, overlapping DNA molecules by the concerted actions of a 5’-exonuclease, a DNA polymerase, and a DNA ligase is described. The DNA fragments are first recessed to produce ssDNA overhangs that are specifically annealed, and then they are covalently joined. This assembly protocol can be used to seamlessly construct synthetic and natural genes, genetic pathways, and entire genomes. This method could be a very useful molecular engineering tool.'The original Gibson assembly protocol is here: http://www.nature.com/protocolexchange/protocols/554The following protocol has minor modifications/optimizations developed that were developed at the Stanford Genome Technology Center (SGTC). They are marked with an asterisk (*) and the reasoning is in italics.
Proper citation: Gergana Vandova 2017. Gibson Assembly. protocols.io dx.doi.org/10.17504/protocols.io.gy5bxy6 Copy
Authors: Fang Wang, Qihan Wang, Vakul Mohanty, Shaoheng Liang, Jinzhuang Dou, Jincheng Han, Darlan Conterno Minussi, Ruli Gao, Li Ding, Nicholas Navin, Ken Chen
Summary: This protocol describes two innovative algorithms:
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1) A minimal event distance aneuploidy lineage tree (MEDALT) inference algorithm allows implementing genetically meaningful distances and is scalable to current single-cell datasets containing thousands of cells, and
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2) A statistical routine, Lineage Speciation Analysis (LSA), enables prioritization of CNAs and genes that are non-randomly associated with the observed lineage expansion and thereby are potentially functionally important.
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Proper citation: Fang Wang, Qihan Wang, Vakul Mohanty, Shaoheng Liang, Jinzhuang Dou, Jincheng Han, Darlan Conterno Minussi, Ruli Gao, Li Ding, Nicholas Navin, Ken Chen 2020. Minimal Event Distance Aneuploidy Lineage Tree (MEDALT) inference based on single cell copy number profile. protocols.io dx.doi.org/10.17504/protocols.io.bfhpjj5n Copy
Authors: Miguel Torres, Tara deBoer
Summary: This protocol is a standard polymerase chain reaction, optimized to amplify a () bp gene flanked by two loxP sites. (?)Refer to page 32, April 5, 2017 entry in Miguel's laboratory notebook for the original document.
Proper citation: Miguel Torres, Tara deBoer 2017. Lox WT gene PCR: amplification from genomic DNA. protocols.io dx.doi.org/10.17504/protocols.io.hnwb5fe Copy
Authors: Isaac Núñez, Tamara Matute
Group: Laboratorio de Tecnologias Libres
Summary: This protocol describes the procedure to obtain single bacterial colonies composed of multiple sectors of different strains. Each sector is originated from different founder cells (situated in close proximity to each other at the moment of plating). This protocol is based on Hallatschek et al. 2007 (Hallatschek O, Hersen P, Ramanathan S, Nelson DR. Genetic drift at expanding frontiers promotes gene segregation. Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):19926-30).This assay has been used for low cost and open source fluorescence imaging (please see https://osf.io/dy6p2/ for further information & data, https://github.com/SynBioUC/FluoPi for code and http://docubricks.com/viewer.jsp? for hardware assembly)
Proper citation: Isaac Núñez, Tamara Matute 2017. Colony-sectoring assay. protocols.io dx.doi.org/10.17504/protocols.io.jsecnbe Copy
Authors: Etienne Laliberté
Group: Canadian Airborne Biodiversity Observatory
Summary: Here we describe the standardised protocol used by the Canadian Airborne Biodiversity Observatory (CABO) to measure leaf spectral reflectance and transmittance, using an integrating sphere fitted to a portable full-range field spectroradiometer, for the special case where an individual leaf is too small and/or too narrow to entirely cover the reflectance or transmission port of the integrating sphere. Briefly, three arrays of mature, healthy and sunlit leaves from a canopy plant are arranged on a custom sample mount, and are then used for measurements of adaxial reflectance and transmittance. Leaf array scans are referenced to a calibrated Spectralon® disk and corrected for stray light to yield NIST-traceable, leaf spectral reflectance and transmittance measurements. Our leaf spectroscopy protocol builds from that of Noda et al. (2013), as well as Carnegie Airborne Observatory's protocol and integrating sphere user manuals from two companies (SVC, ASD Inc.).
Proper citation: Etienne Laliberté 2018. Measuring spectral reflectance and transmittance (350-2500 nm) of small and/or narrow leaves using an integrating sphere. protocols.io dx.doi.org/10.17504/protocols.io.prkdm4w Copy
Authors: Carlos Helbig
Summary: This protocol describes how to transform chemically competent Vibrio natriegens cells.The protocol was described and published by Weinstock et al., 2016
Proper citation: Carlos Helbig 2018. Transformation of Vibrio natriegens. protocols.io dx.doi.org/10.17504/protocols.io.ptsdnne Copy
Authors: Verónica Alejandra Cáceres-Chávez, J. Alejandra Parra-Reyes, Marco A.Herrera-Valdez, Erin Mckiernan
Summary: Patch clamp recording performed in vitro using brain slice preparations is a standard technique used in cellular biophysics and neurophysiology to study the electrical activity of neurons. In particular, our research group is interested in obtaining patch clamp recordings from neurons in the CA1, CA3, and dentate gyrus regions of the hippocampal formation to investigate how the excitability of neurons change during development and aging. To carry out these experiments, we must first dissect out the brain and obtain slices, all while keeping the brain healthy so that the neurons survive and can later be recorded. Here we outline our procedures for anesthetizing, perfusing, dissecting out the brain, and finally obtaining slices. This protocol can also be used as a teaching tool to train students in the handling and dissection of rodents, and the preparation of brain tissue. The slices obtained can also be used for neuroanatomical studies or in training students to identify different brain structures. Our goals in sharing this protocol are to be transparent about our scientific methodology and to help other researchers performing similar experiments.Funding: This work was supported by DGAPA-UNAM-PAPIIT IA209817 and UNAM-DGAPA-PAPIME PE213219 awarded to Erin C. McKiernan. This work was also supported by DGAPA-UNAM-PAPIIT IA208618 and DGAPA-UNAM-PAPIME PE114919 awarded to Marco A. Herrera-Valdez.
Proper citation: Verónica Alejandra Cáceres-Chávez, J. Alejandra Parra-Reyes, Marco A.Herrera-Valdez, Erin Mckiernan 2020. Protocol for obtaining rodent brain slices for electrophysiological recordings or neuroanatomical studies. protocols.io dx.doi.org/10.17504/protocols.io.bggujtww Copy
Authors: Ken Christensen
Summary: SapphireAmp Fast PCR Master Mix contains a hot start PCR enzyme, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent as a 2X premix. SapphireAmp Fast PCR Master Mix is optimized for fast PCR and offers a rapid extension rate (10 sec. per kb). The inclusion of blue dye and a density reagent allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR. SapphireAmp Fast PCR Master Mix is ideal for fast colony PCR screening. Fast colony PCR amplification of a 5 kb insert can be completed in approximately 1 hr 15 min. Furthermore, it is possible to amplify fragments up to 6 kb from genomic DNA templates.
Proper citation: Ken Christensen 2020. SapphireAmp PCR Master Mix -- CHEM 584. protocols.io dx.doi.org/10.17504/protocols.io.bmc7k2zn Copy
Authors: Richard Tennant, Paul Rutten
Group: iGEM Measurement
Summary: You will prepare a dilution series of monodisperse silica microspheres and measure the Abs600 in your plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement will allow you to construct a standard curve of particle concentration which can be used to convert 600 nm absorbance measurements into an estimated equivalent number of cells.
Proper citation: Richard Tennant, Paul Rutten 2019. Calibration Protocol - Particle Standard Curve with Microspheres. protocols.io dx.doi.org/10.17504/protocols.io.zgjf3un Copy
Authors: Max Marrone
Group: Coronavirus Method Development Community, Opentrons COVID-19 Testing
Proper citation: Max Marrone 2020. Operating an OT-2 for COVID-19 testing. protocols.io dx.doi.org/10.17504/protocols.io.bejejcje Copy
Authors: Marijn Ceelen
Group: iGEM Wageningen 2019
Summary: This protocol is used to create concatemers of phage genomes. This is done by ligating phage lambda genomes using the overhangs at the cos site with T4 ligase.
Proper citation: Marijn Ceelen 2019. Phage lambda genome concatemerization. protocols.io dx.doi.org/10.17504/protocols.io.7k3hkyn Copy
Authors: Joshua Timmons
Group: Northeastern iGEM 2015
Summary: This is the PCR protocol for Phusion Polymerase, adapeted from NEB to match the protocol followed by Northeastern_Boston.
Proper citation: Joshua Timmons 2015. PCR with Phusion. protocols.io dx.doi.org/10.17504/protocols.io.dqb5sm Copy
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