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Authors: Joshua Fuller
Summary: A necessary, but difficult, part of ecology and conservations is to try to create order and meaning from chaos. An example of such a task is the need to predict where an organism may exist at any given point in a landscape. In the past, this was done by systematically surveying the entire landscape, a lengthy and expensive endeavor. With the current pace of habitat loss and climate change, new methods were created to efficiently determine the distribution of a species using computers and new statistical algorithms. These new methods still require people in the field taking GPS points of the target species. A way to ease the workload on researchers in the field is to use data generated through citizen science projects such as iNaturalist. Citizen scientists can rapidly generate a tremendous amount of species GPS points by just exploring parks and backyards. That data can be directly utilized by researchers to create a species distribution model (SDM). The goal of this protocol is to take that a step further by generating an SDM for a plant, Tsuga canadensis, and then using that model to determine the distribution of an invasive specialist, Adelges tsugae, on the plant. By the end of this protocol, you should be able to create an SDM, generate a map using GIS software, and critically evaluate the results of the model to determine any conclusions that can be drawn from the model.
Proper citation: Joshua Fuller 2020. Protocol for predicting specialist herbivore distribution from host distribution data. protocols.io dx.doi.org/10.17504/protocols.io.9gvh3w6 Copy
Authors: Steven Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Samantha R Coy's Protocols
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.
Proper citation: Steven Wilhelm 2017. Guava Flow Cytometer - Troubleshooting "Tray Hold Off State". protocols.io dx.doi.org/10.17504/protocols.io.iv9ce96 Copy
Authors: Heidi Fettke
Summary: Circulating tumour DNA (ctDNA), fragmented DNA shed into the circulation from tumour cells, holds the potential to become a minimally-invasive biopsy material with which to interrogate the tumour landscape. However, ctDNA can be highly diluted in normal cell-free DNA (cfDNA) (released into the blood primarily by hematopoietic cells), and thus robust liquid biopsy assays with high analytical sensitivity are required for ctDNA to reach its full clinical potential in molecular genotyping.This protocol uses optimised off-the-shelf reagents in order to generate indexed and molecularly-tagged cfDNA and germline DNA libraries compatible with Illumina short-read sequencing. This protocol involves the hybridisation-capture of targeted regions of the genome using RNA baits - and has been used previously with a 42-gene panel 447 kbp in size. Also described here is the bioinformatic tools used for data processing and variant identification. The combination of deep-sequencing and error-correction based on molecular barcoding allows high sensitivity and specificity to be generated at low variant frequencies.
Proper citation: Heidi Fettke 2020. An error-corrected panel-based next-generation sequencing assay for ultra-sensitive somatic mutation detection in ctDNA. protocols.io dx.doi.org/10.17504/protocols.io.bb3siqne Copy
Authors: Laura Espina
Proper citation: Laura Espina 2020. Preparation of soil bacteria for FCM. protocols.io dx.doi.org/10.17504/protocols.io.bh8qj9vw Copy
Authors: LI-COR Biosciences
Group: LI-COR Biosciences
Summary: Developed for:
Aerius,
Odyssey® Classic,
Odyssey CLx, and
Odyssey Sa
Infrared Imaging Systems
Proper citation: LI-COR Biosciences 2018. Complete Sample Protocol for Measuring IC50 of Inhibitor PD168393 in A431 Cells Responding to Epidermal Growth Factor. protocols.io dx.doi.org/10.17504/protocols.io.grxbv7n Copy
Authors: Shuangshuang Li
Proper citation: Shuangshuang Li 2018. Drug sensitivity assay (CCK8). protocols.io dx.doi.org/10.17504/protocols.io.h4mb8u6 Copy
Authors: Sam Li
Group: BioLegend
Summary: The Anti-Neu5Gc Antibody Kit contains the essential monospecific polyclonal chicken IgY antibody, along with a negative control primary antibody to detect the presence of Neu5Gc on glycoconjugates by Western blot (WB). Samples to be evaluated are first subjected to SDS-PAGE, followed by transfer to a nitrocellulose or polyvinylidenedifluoride (PVDF) membrane. The membrane is then incubated with affinity-purified polyclonal anti-Neu5Gc to determine the presence of Neu5Gc on the protein of interest.The antibody provided in this kit has been shown to identify as little as 5pmol of Neu5Gc per µg glycoprotein, which is at or below the current detection limit for conventional analysis by acid release, purification, DMB derivatization, HPLC, and electrospray mass-spectrometry. The Western blot provides additional information in that it confirms that Neu5Gc is directly linked to the glycoprotein of interest rather than to an accompanying sample component.
Proper citation: Sam Li 2019. Anti-Neu5Gc Antibody Kit Protocol - Western Blot. protocols.io dx.doi.org/10.17504/protocols.io.97hh9j6 Copy
Authors: Andreea S
Group: iGEM Groningen 2020
Summary: This protocol is carried out to observe the growth of the bacterial biofilm along with the roots in the rhizosphere. In the paper by Bennett, R. and Lynch, J (1981), they inoculated the plant with the bacterial strain and analyzed the growth of the bacterial biofilm around roots.
Proper citation: Andreea S 2020. Visualisation of bacteria around roots. protocols.io dx.doi.org/10.17504/protocols.io.bjzpkp5n Copy
Authors: Mark Dewitt, Julia Wong, Beeke Wienert
Group: CornLab
Summary: sgRNA template assembly, in vitro T7 transcription, and sgRNA column cleanup to remove 5'-triphosphate groups
Proper citation: Mark Dewitt, Julia Wong, Beeke Wienert 2020. In vitro transcription of guide RNAs and 5'-triphosphate removal. protocols.io dx.doi.org/10.17504/protocols.io.bfxfjpjn Copy
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 dx.doi.org/10.17504/protocols.io.smeec3e Copy
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 dx.doi.org/10.17504/protocols.io.bkqykvxw Copy
Authors: Lea Starita
Group: Coronavirus Method Development Community
Summary: Molecular Detection of SARS-CoV-2 for SCAN the Greater Seattle Coronavirus Assessment Network
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The SCAN real-time RT-PCR assay for SARS-CoV-2 contains primer/probe sets that target the Orf1b and S genes, designed against the SARS-CoV-2 genome (GenBank: MN908947.3). The primer/probe sets were analyzed in silico for specificity for this specific betacoronavirus by Thermo Fisher. All assays are performed on total nucleic acids extracted from upper respiratory tract swabs, including mid-turbinate and nasopharyngeal (NP) swabs stored in Universal Transport Media. The assay limit of detection of 5.6 (Orf1b) and 12.9 (S) molecules per reaction was determined from serial dilutions of synthetic DNA of the target sequences. These probes reproducibly detected SARS-CoV-2 RNA from cultured virus from USA-WA-1/2020 (BEI resources/ATCC) and from 21 specimens confirmed to be positive by the Washington State Department of health. Specificity was determined by lack of detection of SARS-CoV-2 from specimens that are known to be positive for seasonal coronavirus strains HKU1/NL63 or 229E/OC43 and/or other respiratory viruses. These specimens are further inferred to be negative by the fact that they were obtained in the greater Seattle area prior to the first known reports of SARS-CoV-2 in China.
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Proper citation: Lea Starita 2020. COVID-19 SCAN molecular workflow. protocols.io dx.doi.org/10.17504/protocols.io.bebkjakw Copy
Authors: Ken Stedman, Geoffrey Diemer, Jennifer E Kyle
Group: VERVE Net
Summary: An alternative epifluorescence microscopy (EFM) technique for virus enumeration has been developed using polycarbonate Track Etch (PCTE) filter membranes in place of aluminum oxide Anodisc membranes. The established EFM technique, first developed by Hennes and Suttle in 1995 and later expanded upon by Noble and Fuhrman in 1998, uses supported 20 nm pore-size Anodisc filter membranes to determine virus abundance in natural environments. Increased price and sporadic availability of Anodisc filters stimulated the evaluation of alternative filters for use in the procedure. The feasibility of using 30 nm pore-size PCTE filters for virus enumeration was assessed using the Anodisc filter procedure as a control. Although virion particle counts are slightly less precise using PCTE filters, they offer a substitute for Anodiscs while requiring only minor adjustments to the established protocol. Per slide, the PCTE costs approximately ten times less to prepare than the Anodisc-based method.
Proper citation: Ken Stedman, Geoffrey Diemer, Jennifer E Kyle 2016. PCTE Protocol for VLP enumeration. protocols.io dx.doi.org/10.17504/protocols.io.eaubaew Copy
Authors: James Skelton, Craig Bateman, Jiri Hulcr
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol describes how to extract ambrosia fungi from Xylosandrus compactus, the black twig borer. This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.
Proper citation: James Skelton, Craig Bateman, Jiri Hulcr 2020. Fungal extraction from Xylosandrus compactus. protocols.io dx.doi.org/10.17504/protocols.io.bnuumeww Copy
Authors: Jacob Harris
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Alverson Lab
Summary: The FlowCam can be used to take quick cell density measurements of liquid samples.
Proper citation: Jacob Harris 2016. FlowCam Standard Operating Procedure. protocols.io dx.doi.org/10.17504/protocols.io.f2tbqen Copy
Authors: Grossman Lab, Pringle Lab
Group: Aiptasia-Symbiodiniaceae Model System
Summary: This protocol describes briefly how to perform colonization of aposymbiotic Aiptasia with Symbiodinium cultures.The method is based on the one described in Xiang et al., 2013.
Proper citation: Grossman Lab, Pringle Lab 2018. Colonization of aposymbiotic Aiptasia with Symbiodinium. protocols.io dx.doi.org/10.17504/protocols.io.rh4d38w Copy
Authors: James Fraser
Group: Fraser Lab
Proper citation: James Fraser 2015. QuikChange Mutagenesis. protocols.io dx.doi.org/10.17504/protocols.io.dij4cm Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes PCR1 and PCR2 reactions beginning with cDNA to generate transcript templates ready for IVT reaction. This protocol is written for 192 (2x96) reactions but can also be used for fewer or more reactions by adjusting the amount of materials and equipment appropriately.
Proper citation: Allen Institute for Brain Science 2020. PCR Prep from cDNA for IVT Reactions. protocols.io dx.doi.org/10.17504/protocols.io.bcsfiwbn Copy
Authors: Soham Adla, Neeraj Rai, K Sri Harsha, Shivam Tripathi, Markus Disse, Saket Pande
Proper citation: Soham Adla, Neeraj Rai, K Sri Harsha, Shivam Tripathi, Markus Disse, Saket Pande 2018. Laboratory calibration of soil moisture sensors in porous media (repacked soils). protocols.io dx.doi.org/10.17504/protocols.io.pjidkke Copy
Authors: Elizabeth Jannaman, M. Sofia Ortega, Tad S. Sonstegard, Peter Hansen
Group: University of Florida Bovine Embryo Lab
Summary: Several mutations exist in the prolactin receptor gene (PRLR) of cattle that cause a short, sleek haircoat.. The mutation is dominant so slick-haired cattle can have either one ortwo copies of the mutation. The assay described here detects the most-well studied mutation (SLICK1)that causes translation of a truncated PRLR of 461 amino acids. Originally described in Senepol cattle,this mutation has also been introduced into Holsteins in Puerto Rico, Florida and elsewhere. Note thatthe assay will not detect other mutations in PRLR that also cause the slick phenotype.
Proper citation: Elizabeth Jannaman, M. Sofia Ortega, Tad S. Sonstegard, Peter Hansen 2019. PCR-based assay for genotyping of the slick mutation in cattle. protocols.io dx.doi.org/10.17504/protocols.io.6kxhcxn Copy
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