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Authors: James Lloyd, Ryan Lister
Summary: To extract plasmid DNA from Agrobacterium, to confirm the correct plasmid has been taken-up, this protocol allows you to directly perform a mini-prep on transformed Agrobacterium, digest this DNA and visualize it on a gel. This is without the need of rescuing the plasmid in E. coli or genotyping with PCR. This protocol using the commercially available Bioline ISOLATE II Plasmid Mini Kit, designed for use with E. coli. The standard protocol this is modified from is available here: https://www.bioline.com/au/downloads/dl/file/id/1219/isolate_ii_plasmid_mini_kit_protocol.pdf
Proper citation: James Lloyd, Ryan Lister 2018. Extracting plasmid DNA from Agrobacterium with the Bioline ISOLATE II Plasmid Mini Kit. protocols.io dx.doi.org/10.17504/protocols.io.s8fehtn Copy
Authors: Angel Justiz-Vaillant, Norma McFarlane-Anderson
Group: University of the West Indies, [email protected]
Proper citation: Angel Justiz-Vaillant, Norma McFarlane-Anderson 2020. Universal sandwich enzyme linked immunosorbent assay for investigating Streptococcal protein-G (SpG) interactions with immunoglobulins using a SpLA-HRP conjugate.. protocols.io dx.doi.org/10.17504/protocols.io.bjpvkmn6 Copy
Authors: Mark Moosburner
Group: A.E. Allen Lab
Summary: I am creating this protocol to deposit a plasmid sequence and map. The episome contains Cas9-2A-ShBle expression cassette and 2 sgRNA expression cassettes targeting the gene Phatr3_J24739.
Proper citation: Mark Moosburner 2019. Pt_Cas9-2A-ShBle, g24739-A, mRFP, g24739-B. protocols.io dx.doi.org/10.17504/protocols.io.7gnhjve Copy
Authors: Itay Barnea
Summary: Sperm isolation and fixation
Proper citation: Itay Barnea 2017. Sperm isolation and fixation. protocols.io dx.doi.org/10.17504/protocols.io.me7c3hn Copy
Authors: Leila Pishevar
Proper citation: Leila Pishevar 2017. Effect of food simulating liquids on the flexural strength of the methacrylate and silorane-based composites. protocols.io dx.doi.org/10.17504/protocols.io.icbcasn Copy
Authors: Zita Santos, Patrícia Francisco, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro
Group: Ribeiro Lab
Summary: This is a collection of methods and protocols from the manuscript: Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.
Proper citation: Zita Santos, Patrícia Francisco, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro 2017. Methods and protocols from Goncalves et al. (2017) for manipulating the diet and the microbiome of Drosophila. protocols.io dx.doi.org/10.17504/protocols.io.hdtb26n Copy
Authors: Snehadri Sinha
Group: Aalto-Helsinki 2016
Summary: PCR using Phusion HotStart II Hifi Mastermix (based on ThermoFisher Scientific manufacturer protocol).
Proper citation: Snehadri Sinha 2017. Phusion HotStart II Hifi PCR. protocols.io dx.doi.org/10.17504/protocols.io.gzjbx4n Copy
Authors: Bei Wang, Daniel Asarnow, Wen-Hsin Lee, Ching-Wen Huang, Bryan Faust, Patricia Miang Lon Ng, Eve Zi Xian Ngoh, Markus Bohn, David Bulkley, Andrés Pizzorno, Hwee Ching Tan, Chia-Yin Lee, Rabiatul Adawiyah Minhat, Olivier Terrier, Mun Kuen Soh, Frannie Jiuyi Teo, Yvonne Yee Chin Yeap, Yuanyu Hu, Shirley Gek Kheng Seah, Sebastian Maurer-Stroh, Laurent Renia, Brendon John Hanson, Manuel Rosa-Calatrava, Aashish Manglik, Yifan Cheng, Charles S. Craik, Cheng-I Wang
Group: Coronavirus Method Development Community
Summary: In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the “up” and the other in the “down” position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.
Proper citation: Bei Wang, Daniel Asarnow, Wen-Hsin Lee, Ching-Wen Huang, Bryan Faust, Patricia Miang Lon Ng, Eve Zi Xian Ngoh, Markus Bohn, David Bulkley, Andrés Pizzorno, Hwee Ching Tan, Chia-Yin Lee, Rabiatul Adawiyah Minhat, Olivier Terrier, Mun Kuen Soh, Frannie Jiuyi Teo, Yvonne Yee Chin Yeap, Yuanyu Hu, Shirley Gek Kheng Seah, Sebastian Maurer-Stroh, Laurent Renia, Brendon John Hanson, Manuel Rosa-Calatrava, Aashish Manglik, Yifan Cheng, Charles S. Craik, Cheng-I Wang 2020. Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization. protocols.io dx.doi.org/10.17504/protocols.io.bi35kgq6 Copy
Authors: Peter Vangheluwe, Shaun Martin, Mujahid Azfar
Summary: The 4-methylumbelliferyl heptanoate (MUH) assay is a A fluorimetric assay used to study cell-mediated cytotoxicity. The principle behind this assay employs the activity of esterases found in viable cells. These esterases then hydolyse the fluorochrome in the MUH reagent which can then be read using a plate reader.
Proper citation: Peter Vangheluwe, Shaun Martin, Mujahid Azfar 2020. 4-methylumbelliferyl heptanoate (MUH) - Cell Viability Assay. protocols.io dx.doi.org/10.17504/protocols.io.bazjif4n Copy
Authors: Keyong Sun
Group: LanXun_lab
Proper citation: Keyong Sun 2020. Blunt end ligation. protocols.io dx.doi.org/10.17504/protocols.io.bdywi7xe Copy
Authors: Antonio Hartley
Proper citation: Antonio Hartley 2018. Inmunohistoquímica rápida. protocols.io dx.doi.org/10.17504/protocols.io.r7sd9ne Copy
Authors: Barbora Lubyova
Proper citation: Barbora Lubyova 2017. PRMT5: a Novel Regulator of Hepatitis B Virus Replication and an Arginine Methylase of HBV Core. protocols.io dx.doi.org/10.17504/protocols.io.je8cjhw Copy
Authors: Zita Santos, Patrícia Francisco, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro
Group: Ribeiro Lab
Summary: This is a protocol to generate axenic fly cultures by sterilizing embryos. It is part of the manuscript: Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.The protocol was adapted from:Wayland MT, Defaye A, Rocha J, Jayaram SA, Royet J, Miguel-Aliaga I, et al. Spotting the differences: Probing host/microbiota interactions with a dedicated software tool for the analysis of faecal outputs in Drosophila. J Insect Physiol. 2014;69: 126–135. doi:10.1016/j.jinsphys.2014.05.023with the help of the Leulier laboratory.
Proper citation: Zita Santos, Patrícia Francisco, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro 2017. Generating and Rearing Axenic Drosophila. protocols.io dx.doi.org/10.17504/protocols.io.hebb3an Copy
Authors: Alyssa Alsante
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Proper citation: Alyssa Alsante 2016. MBBM Media. protocols.io dx.doi.org/10.17504/protocols.io.f3dbqi6 Copy
Authors: Daphna Strauss, Ayelet Rahat, Israa Sharkia, Alon Chappleboim, Miriam Adam, Daniel Kitsberg, Gavriel Fialkoff, Matan Lotem, Omer Gershon, Anna-Kristina Schmidtner, Esther Oiknine-Djian, Agnes Klochendler, Ronen Sadeh, Yuval Dor, Dana Wolf, Naomi Habib, Nir Friedman
Summary: The global SARS-CoV-2 pandemic led to a steep increase in the need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays that involve a separate RNA extraction and qPCR reaction for each sample with a fixed cost and reaction time. While automation and improved logistics can increase the capacity of these tests, they cannot exceed this lower bound dictated by one extraction and reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information on viral strains and host immune response.Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, ApharSeq (Amplicon Pooling by Hybridization And RNA-Seq), includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one step is performed before pooling hundreds of barcoded samples for subsequent steps and further analysis. Considering these improvements, our proposed workflow is estimated to reduce costs by 10-50 fold, labor by 5-100 fold, automated liquid handling by 5-10 fold, and reagent requirements by 100-1000 fold compared to existing methods.
Proper citation: Daphna Strauss, Ayelet Rahat, Israa Sharkia, Alon Chappleboim, Miriam Adam, Daniel Kitsberg, Gavriel Fialkoff, Matan Lotem, Omer Gershon, Anna-Kristina Schmidtner, Esther Oiknine-Djian, Agnes Klochendler, Ronen Sadeh, Yuval Dor, Dana Wolf, Naomi Habib, Nir Friedman 2020. SARS-CoV-2 detection with ApharSeq. protocols.io dx.doi.org/10.17504/protocols.io.bjgukjww Copy
Authors: Sarah Reiling, Anne-Marie Roy, Shu-Huang Chen, Ioannis Ragoussis
Group: McGill Genome Centre
Summary: This protocol works for 12 native barcodes, 24 native barcodes, and 96 native barcodes.
Proper citation: Sarah Reiling, Anne-Marie Roy, Shu-Huang Chen, Ioannis Ragoussis 2020. McGill Nanopore Native Barcoding LibPrep Protocol,10 ng NB . protocols.io dx.doi.org/10.17504/protocols.io.bmijk4cn Copy
Authors: Elizabeth Butterworth, Wesley Dickerson, Vindhya Vijay, Kristina Weitzel, Julia Cooper, Eric W. Atkinson, Jason E. Coleman, Kevin Otto, Martha Campbell Thompson
Group: Human Cell Atlas Method Development Community, Optical Clearing of Tissue, SPARC
Summary: Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to 500 μm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 μm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.
Proper citation: Elizabeth Butterworth, Wesley Dickerson, Vindhya Vijay, Kristina Weitzel, Julia Cooper, Eric W. Atkinson, Jason E. Coleman, Kevin Otto, Martha Campbell Thompson 2020. Human Pancreas PACT Optical Clearing and High Resolution 3D Microscopy. protocols.io dx.doi.org/10.17504/protocols.io.9gbh3sn Copy
Authors: Alle Lie
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology
Proper citation: Alle Lie 2016. Making heat-killed bacteria for feeding axenic phagotrophic protists. protocols.io dx.doi.org/10.17504/protocols.io.ewkbfcw Copy
Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: Target cells are positively selected or depleted by incubating the sample with an anti-human APC conjugated antibody, followed by incubation with magnetic anti-APC Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. These are the APC+ cells, do not discard them if those are the cells of interest. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.
Proper citation: Sam Li 2019. MojoSort™ Mouse anti-APC Nanobeads Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7xhhpj6 Copy
Authors: ruangp
Proper citation: ruangp 2020. Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits. protocols.io dx.doi.org/10.17504/protocols.io.bpyrmpv6 Copy
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