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Authors: Alba Balletbó
Group: iGEM Wageningen 2019
Summary: Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer ones. Thus, you can determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths).

Proper citation: Alba Balletbó 2019. Gel Electrophoresis. protocols.io https://dx.doi.org/10.17504/protocols.io.7eehjbe Copy   

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Authors: NEB
Group: New England Biolabs (NEB)

Proper citation: NEB 2016. Digestion Mixture for M0302 (v2). protocols.io https://dx.doi.org/10.17504/protocols.io.erhbd36 Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Phenotyping of Rodents for the Presence of Diabetic Neuropathy In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.Diabetic Complication:

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Immunohistochemistry for Tissue Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.7rfhm3n Copy   

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Authors: Honghao Zhao, Jasmine Chong, Rong Tang, Li Li, Jianguo Xia, Dapeng Li
Summary: This protocol is widely used in serum biochemical indexes detection, it provides an rapid and reliable technique for obtaining relative concentrations of multiple blood biochemical indices simultaneously.

Proper citation: Honghao Zhao, Jasmine Chong, Rong Tang, Li Li, Jianguo Xia, Dapeng Li 2018. Serum Biochemical Indexes Detection. protocols.io https://dx.doi.org/10.17504/protocols.io.smeec3e Copy   

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Authors: Fatma Gomaa, Paulo Garcia, Jennifer Delany, Cullen Buie, Peter Girguis, and Virginia Edgcomb

Proper citation: Fatma Gomaa, Paulo Garcia, Jennifer Delany, Cullen Buie, Peter Girguis, and Virginia Edgcomb 2017. Transient genetic transformation of Bodo caudatus using square wave electroporation system and pUB-GFP plasmid. protocols.io https://dx.doi.org/10.17504/protocols.io.g5yby7w Copy   

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Authors: Izabela Rezende, Lívia Sacchetto
Group: MRCA
Summary: For purification of viral RNA from serum and cell-culture supernatants.

Proper citation: Izabela Rezende, Lívia Sacchetto 2019. Total RNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.pw7dphn Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The BioBrick® Assembly Kit was developed in partnership with Ginkgo BioWorks. What follows is an abbreviated set of protocols for the use of the BioBrick® Assembly Kit (to assemble an Upstream Part with a Downstream Part into a Destination Plasmid). For more details and for technical questions, please see here.

Proper citation: New England Biolabs 2015. Digestion for BioBrick Assembly Kit (E0546). protocols.io https://dx.doi.org/10.17504/protocols.io.cphvj5 Copy   

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Authors: Sureshnee Pillay, Jennifer Giandhari, Houriiyah Tegally, Eduan Wilkinson, Benjamin Chimukangara, Richard Lessells, Yunus Moosa, Inbal Gazy, Maryam Fish, Lavanya Singh, Khulekani Sedwell Khanyile, Vagner Fonseca, Marta Giovanetti, Luiz Carols Alcantara, Tulio de Oliveira, Jennifer Giandhari
Group: Coronavirus Method Development Community, KRISP
Summary: This protocol describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing amplicons tiling the viral genome sequencing. It uses the V3 nCov-2019 primers from the ARTIC network. This is followed by library construction using Nextera Flex, which we found to save 9h of hands on time as compared with original protocol that use TrueSeq for library contstruction. It also describes the pooling of samples and quantitation, prior to sequencing on the Illumina Miseq and NextSeq. It is adapted from the nCov-2019 sequencing protocol from Quick and colleagues, which can be found here:

Proper citation: Sureshnee Pillay, Jennifer Giandhari, Houriiyah Tegally, Eduan Wilkinson, Benjamin Chimukangara, Richard Lessells, Yunus Moosa, Inbal Gazy, Maryam Fish, Lavanya Singh, Khulekani Sedwell Khanyile, Vagner Fonseca, Marta Giovanetti, Luiz Carols Alcantara, Tulio de Oliveira, Jennifer Giandhari 2020. Illumina Nextera DNA Flex library construction and sequencing for SARS-CoV-2: Adapting COVID-19 ARTIC protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bhjgj4jw Copy   

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Authors: Steven Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.

Proper citation: Steven Wilhelm 2017. Fluorescent Staining of T5-phages. protocols.io https://dx.doi.org/10.17504/protocols.io.iksccwe Copy   

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Authors: Chi-Kuang Yao, Yu-Tzu Liu, I-Chi Lee, You-Tung Wang, Ping-Yen Wu
Summary: This protocol describes the generation of mFwe Knockout n2a cells. It is from 'Flower Ca2+ channel in CME and ADBE' of Yao CK et al. Please see the manuscript for the full method details.

Proper citation: Chi-Kuang Yao, Yu-Tzu Liu, I-Chi Lee, You-Tung Wang, Ping-Yen Wu 2017. Generation of mFwe knockout n2a cells from Yao CK et al. (2017). protocols.io https://dx.doi.org/10.17504/protocols.io.hgwb3xe Copy   

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Authors: Elizabeth Fozo

Proper citation: Elizabeth Fozo 2020. 3. LiCor_Blotting_Protocol. protocols.io https:// Copy   

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Authors: Lora Starrs, Debra Brown, Sarah Popp, Andrew Ziolkowski, Charmaine Simeonovic
Summary: Paraffin sections (4 μm thickness) of formalin-fixed human pancreases and isolated human islets were treated with 0.05% pronase for antigen retrieval. HS and Col18 HSPG core proteins were detected immunohistochemically using 10E4 anti-HS (US Biological/Amsbio) and anti-Col18 (Santa Cruz), respectively, with horseradish peroxidase-conjugated rabbit anti-mouse 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, Andrew Ziolkowski, Charmaine Simeonovic 2020. Immunohistochemical staining of heparan sulfate (HS) and collagen type XVIII (col18) core proteins in islet beta cells of formalin-fixed human pancreas and isolated human islets. protocols.io https://dx.doi.org/10.17504/protocols.io.bkqykvxw Copy   

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Authors: Theresa Pollinger
Summary: https://wickie.hlrs.de/platforms/index.php/Cray_XC40

Proper citation: Theresa Pollinger 2018. Using HLRS Hazel Hen supercomputer. protocols.io https://dx.doi.org/10.17504/protocols.io.ud8es9w Copy   

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Authors: Christopher Willey
Group: NCI PDMC consortium

Proper citation: Christopher Willey 2020. UABMC - Stemness Determination Protocol. protocols.io https:// Copy   

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Authors: Larry Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Larry Klobutcher 2017. Growth of Dunaliella salina on artificial seawater.. protocols.io https://dx.doi.org/10.17504/protocols.io.gwpbxdn Copy   

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Authors: Ian Simpson
Group: Diabetic Complications Consortium
Summary: This document describes the procedure for inducing a stroke in diabetic RCS10 mice, their non-diabetic controls and NON/ShiLtJ control mice. Our initial objective was to use those RCS10 mice that did not become diabetic as the controls for the experiment to compare the effect of diabetes on stroke outcome and that data is illustrated in Figure 2. However, with further breading the numbers of non- diabetic RCS 10 mice at twenty weeks of age were so few that it became necessary to use the NON/ShiLtJ mice which are 85% genetically identical to the RCS10 mice and stroke outcome is illustrated in Figure 3.Diabetic Complications:

Proper citation: Ian Simpson 2019. Induce Stroke via Hypoxia-Ischemia. protocols.io https://dx.doi.org/10.17504/protocols.io.8e5htg6 Copy   

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Authors: Ken Christensen
Summary: Product DescriptionThe Zymoclean™ Gel DNA Recovery Kit provides a hassle-free method for high yield recovery of pure DNA from agarose gels. Simply add the specially formulated Agarose Dissolving Buffer (ADB) to the gel slice containing your DNA sample, let dissolve, and then transfer to the supplied Zymo-Spin™ Column. There is no need for organic denaturants or chloroform. Instead, the product utilizes Fast-Spin column technology to yield high-quality DNA in just 15 minutes. DNApurified using the Zymoclean™ Gel DNA Recovery Kit is perfectly suited for use in DNA ligation reactions, sequencing, DNA labeling reactions, PCR, etc.

Proper citation: Ken Christensen 2020. Zymoclean Gel DNA Recovery--CHEM 584. protocols.io https://dx.doi.org/10.17504/protocols.io.bmyzk7x6 Copy   

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Authors: Sebastian Poljak
Summary: We report a phylogeographic study of Chaetophractus villosus populations in Argentina. Control Region (CR) sequences (484 bp) were obtained for 76 C. villosus from 20 locations across the species whole distribution range. Seventeen new haplotypes were identified. The highest genetic variation and the earliest fossils were found in the Pampean Region, thus appearing as the most probable area of origin of the species. A general pattern of Contiguous Range Expansion (CRE) was revealed by Nested Clade Analysis (NCA) supported by mismatch analysis and Fu’s test. The Pampean Region would have been the preexpansion area, while Patagonia would have been the main dispersal route of contiguous expansion, possibly after the Pleistocenic glaciations.

Proper citation: Sebastian Poljak 2017. PCR partial Control Region Chaetophractus 456 bp. protocols.io https://dx.doi.org/10.17504/protocols.io.kmncu5e Copy   

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Authors: Alise Ponsero, Bonnie Hurwitz
Group: Metafunc course 2018
Summary: This protocol explores how to run USEARCH (v11.0.667) (Edgar, 2010) on a 16s RNA dataset.USEARCH is a sequence analysis tool that offers search and clustering algorithms. Here USEARCH will be used to run a quality control on a 16S RNA dataset. This protocol uses a staggered mock community as an example (see attached files).

Proper citation: Alise Ponsero, Bonnie Hurwitz 2018. Run USEARCH for quality control of a 16s RNA dataset. protocols.io https://dx.doi.org/10.17504/protocols.io.s8iehue Copy   

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Authors: Integrated Islet Distribution Program
Group: Integrated Islet Distribution Program
Summary: This SOP defines the pancreas donor profile acceptable for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP). This SOP applies to all IIDP islet distribution centers using funds from the NIDDK to manufacture purified human pancreatic islets for basic research studies for IIDP approved investigators.The IIDP depends on the subcontracted human islet isolation centers to provide research investigators with human islets. It is the responsiblity of the human islet centers to obtain research quality pancreata from the Organ Procurement Organizations (OPO). Some criteria are stricter than those used by transplant centers for organ transplant donors. This protocol provides the inclusion and exclusion criteria in order to determine the proper parameters for research donors.The donor criteria also reflects the required 2020 testing for COVID-19. Positive testing for COVID-19, HIV, Hepatitis C, and Hepatitis B are examples of some exclusion criteria.References (attached)OPTN Policies Effective as of April 3 2020 [Emergency COVID-19 Policy Actions]Organ Procurement and Transplantation Network, 2020 Apr.2013 PHS Guideline for Reducing Human Immunodeficiency Virus, Hepatitis B Virus, and Hepatitis C Virus Transmission through Organ Transplantation1 Behavioral Risk Factors for Recent HIV, HBV, or HCV Infection. Organ Procurement and Transplantation Network, 2013 Nov.

Proper citation: Integrated Islet Distribution Program 2020. Determining IIDP Minimal Donor Criteria. protocols.io https://dx.doi.org/10.17504/protocols.io.bfrpjm5n Copy   

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