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Authors: Jongeun Park
Group: Human Cell Atlas Method Development Community
Summary: This is protocol for fetal thymus dissociation. (Can be applied to other fetal tissues)Can be also adapted to adult thymus or lymphoid organs. (Volume of solution needs to be adjusted to take account of larger tissue size, also consider multiple rounds of digestion in this case)

Proper citation: Jongeun Park 2018. Cambridge Fetal Thymus Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.wedfba6 Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab

Proper citation: Matthew Sullivan 2016. Host Range Protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.dew3fd Copy   

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Authors: Rengin Yamur Akbiyik, Sevval Uysalcan
Summary: Transformation is a direct modification of the genotype of a cell from a different one by extracellular applications using recombinant DNA techniques. Transformation refers generally to the integration of exogenous DNA into the cell and its integration into the genome. Before starting the transformation process, the gene of interest is ligated with the plasmid vector via the ligase enzyme. This process is called ligation. Generally, nucleic acids or plasmids cannot enter into bacterial cells by themselves. The stimulatory effect is required for that purpose. This means that the cell membranes to be transformed must be pre-arranged. Bacteria that can contain free DNA are called competent bacteria. Some bacteria are highly competitive in normal growth conditions; but some must be treated with chemical or physical methods to gain competitive properties. Competent cells which are cells are generally stimulated with calcium chloride about chemically and the cell membranes are arranged such that the plasmid vector containing the gene of interest can harbor the vector.Some bacterial cells are lasting competent, however; most of them need to be influenced forbeing competent. There are two ways about this transformation process that;• Chemical Transformation• Electrical Transformation (Electroporation)In general, chemical transformation is used. In chemical way, some special chemicals are used to open the pores which are found in cell membrane. This is because, opening the pores represents the availability of being permeable. Most of the time, divalent ions like Ca++ are used with assistance of heat-shock.

Proper citation: Rengin Yamur Akbiyik, Sevval Uysalcan 2019. Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.2nwgdfe Copy   

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Authors: Lora Starrs, Debra Brown, Sarah Popp, Charmaine Simeonovic
Summary: Paraffin sections (4 μm thickness) of formalin-fixed human pancreases were treated with heat/citrate buffer for antigen retrieval. Sdc-1core proteins were detected immunohistochemically using rat anti-mouse CD138 (Sdc-1) mAb (BD Biosciences), with horseradish peroxidase-conjugated rabbit anti-rat Ig (Dako). Background staining was checked using the corresponding isotype control Ig instead of the primary antibody. 3-amino-9-ethylcarbazole (AEC) was used as the chromogen. For morphometry, stained sections were imaged using a light microscope with attached camera (Olympus BX41). Image J software with color deconvolution plugin was used for the quantitative analysis of the % of islet area stained.

Proper citation: Lora Starrs, Debra Brown, Sarah Popp, Charmaine Simeonovic 2020. Immunohistochemical staining of Syndecan-1 (Sdc-1) core proteins in islet beta cells of formalin-fixed human pancreas. protocols.io https://dx.doi.org/10.17504/protocols.io.bkqxkvxn Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Cas9 Nuclease, S. pyogenes (Cas9), is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA in vitro using Cas9 and a single guide RNA (sgRNA).

Proper citation: New England Biolabs 2014. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386). protocols.io https://dx.doi.org/10.17504/protocols.io.ch2t8d Copy   

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Authors: Binnypreet Kaur
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes
Summary: Cas9 Nuclease, S. pyogenes, (Cas9) is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA in vitro using Cas9 and a single guide RNA (sgRNA). 

Proper citation: Binnypreet Kaur 2018. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386). protocols.io https://dx.doi.org/10.17504/protocols.io.rmud46w Copy   

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Authors: Dima Ter-Ovanesyan, Wendy Trieu , Maia Norman, Roey Lazarovits, George Church, David Walt
Group: Walt Lab
Summary: Extracellular vesicles (EVs) are released by all mammalian cells and are thought to be important mediators ofintercellular communication. There are many methods for isolating EVs from cell culture media, but one of themost commonly used methods continues to be purification based on ultracentrifugation. This approach has the advantage of allowing a large input volume of cell culture media. Here, we provide a detailedprotocol for isolating EVs by differential ultracentrifugation.

Proper citation: Dima Ter-Ovanesyan, Wendy Trieu , Maia Norman, Roey Lazarovits, George Church, David Walt 2020. Isolation of Extracellular Vesicles from Cell Culture Media by Differential Ultracentrifugation . protocols.io https://dx.doi.org/10.17504/protocols.io.bnr3md8n Copy   

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Authors: Yasser Amer, Turki Albatti, Fahad Bashiri, Muddathir Hamad, Haya Al-Joudi, Saleh Al Salehi, Hadeel Daghash, Jeremy Varnham
Summary: PROSPERO protocolLink: http://www.crd.york.ac.uk/PROSPERO/display_record.php?ID=CRD42017078712

Proper citation: Yasser Amer, Turki Albatti, Fahad Bashiri, Muddathir Hamad, Haya Al-Joudi, Saleh Al Salehi, Hadeel Daghash, Jeremy Varnham 2018. Appraisal of clinical practice guidelines for the management of attention deficithyperactivity disorder (ADHD) using the AGREE II Instrument: a systematic review. protocols.io https://dx.doi.org/10.17504/protocols.io.q27dyhn Copy   

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Authors: Maysa Silva, Maryana Branquinho, Maria Cármen Sales

Proper citation: Maysa Silva, Maryana Branquinho, Maria Cármen Sales 2018. RNA extraction with trizol for tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.stmeek6 Copy   

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Authors: David Frommholz, Nadine Stefanczyk, Alexandra Ehl
Summary: Purification Guide for the Isolation of Histidine-tagged Proteins with ChroDrip Columns by DALEX Biotech.ChroSpin-IMAC by DALEX Biotech offers a robust and convenient way to isolate polyhistidine-tagged protein from bacterial, mamalian, and insect cell cultures.Immobilized metal affinity chromatography (IMAC) is based on the interaction of the imidazole ring of histidine with transition metal ions immobilized on a solid support. Recombinant proteins with a 3 - 10 histidine fusion tag bind to these metal ions while unwanted proteins are removed by washing with excess binding buffer. Elution of the target protein is achieved by the addition of imidazole, EDTA or a low pH. Easy and quick small scale fusion protein purification from various sources.Low metal leaching from column.Tolerates reducing reagents (e.g. DTT up to 10 mM).The proprietary resin does not shrink or swell in aqueous buffers.High pressure stability.pH stability short term 2 - 8, long term 3 - 8.Excellent thermal stability up to 15 minutes at 120 °C in aqueous buffers at neutral pH.Can be dried for long term storage (80 °C for > 2 h).

Proper citation: David Frommholz, Nadine Stefanczyk, Alexandra Ehl 2018. ChroDrip - IMAC. protocols.io https://dx.doi.org/10.17504/protocols.io.uvvew66 Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural rosette banking. protocols.io https://dx.doi.org/10.17504/protocols.io.x87frzn Copy   

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Authors: Jackie L. Collier
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: Modified from Lippmeier et al. 2009

Proper citation: Jackie L. Collier 2018. Labyrinthulomycete DNA extraction protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.n83dhyn Copy   

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Authors: Felix Kraus, vtrinkaus , Ruben Fernandez Busnadiego
Summary: This protocol details methods for correlative light-electron microscopy of α-synuclein aggregates in primary neurons.

Proper citation: Felix Kraus, vtrinkaus , Ruben Fernandez Busnadiego 2021. Correlative Light-Electron Microscopy of α-synuclein Aggregates in Primary Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.btyinpue Copy   

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Authors: Kenneth Schackart
Group: Yoon Lab
Summary: How to run nucleic acid amplification using the Thermo scientific PCR Master Mix kit.Each reaction produces 50 μL.For the original protocol, look at: .

Proper citation: Kenneth Schackart 2019. PCR Reaction Optimization. protocols.io https://dx.doi.org/10.17504/protocols.io.ygqftvw Copy   

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Authors: Shuntai Zhou
Summary: This is the protocol to prepare Primer ID MiSeq sequencing library. Viral RNA was first extracted using QIAamp viral RNA extraction kit.  The block of random nucleotides (Ns) in the cDNA primers served as the Primer ID. The Superscript III kit was used for the cDNA synthesis. We used two rounds of PCR to amplify the cDNA and incorporate Illumina indexed adapters with KAPA2G Robust and KAPA HiFi PCR kits, respectively. 

Proper citation: Shuntai Zhou 2019. Primer ID MiSeq Library Prep for HIV-1 DR and diversity. protocols.io https://dx.doi.org/10.17504/protocols.io.6hchb2w Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)

Proper citation: New England Biolabs 2020. Protocol for Exonuclease III (NEB #M0206). protocols.io https:// Copy   

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Authors: Gloria Pryhuber, Heidie Huyck, Lisa Rogers, Cory Poole
Group: Human Cell Atlas Method Development Community, Human BioMolecular Atlas Program (HuBMAP) Method Development Community, LungMap2 Consortium, URMC Pryhuber Lab
Summary: Purpose and Scope of the Procedure- Rapid blocking, embedding and freezing of human lung tissue in no freezing media, 100% OCT or 5% CMC - Rapid freezing of non-lung tissue in no freezing media, 100% OCT or 5% CMC

Proper citation: Gloria Pryhuber, Heidie Huyck, Lisa Rogers, Cory Poole 2020. 615.1 URMC HTC Non-Inflated Fresh-Frozen Embedded Lung and Associated Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bjtnknme Copy   

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Authors: Chad Vezina

Proper citation: Chad Vezina 2020. Vezina Lab Mouse Kidney Capsule Implant Protocol. protocols.io https:// Copy   

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Authors: Niels Appelman
Group: iGEM Wageningen 2019
Summary: This protocol is used to capture spittlebugs (Philaenus spumarius) and Green Leafhoppers (Cicadella viridis)

Proper citation: Niels Appelman 2019. Capture of Philaenus spumarius and Cicadella viridis. protocols.io https://dx.doi.org/10.17504/protocols.io.8jdhui6 Copy   

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Authors: Ken Youens-Clark
Group: MetaFunc Course
Summary: This describes setting up ssh keys and configuration to avoid having to use your NetID+ 2-factor authentication every time you log into the HPC.  Windows users are encouraged to install Cygwin (http://www.cygwin.com), a free Unix-like environment that provides a terminal so as to avoid using an ssh client like Putty.  Cygwin users must be sure to install the "ssh" tools which are not installed by default.

Proper citation: Ken Youens-Clark 2016. ssh to UA HPC. protocols.io https://dx.doi.org/10.17504/protocols.io.fm7bk9n Copy   

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