Protocols   Select Another Resource Report Type

Authors: Benjamin Schwessinger and Megan McDonald
Group: High molecular weight DNA extraction from all kingdoms
Summary: Extraction of high quality DNA for long read sequencing e.g. PacBioOptimized for DNA extraction from wheat stripe rust spores and also tested on barley leaf rust.Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.Critical steps to obtain high quality DNA:Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.Do NOT incubate samples with KAc for prolonged time periodsPerform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction. Summary statistics of sequencing runs to follow.

Proper citation: Benjamin Schwessinger and Megan McDonald 2017. High quality DNA from Fungi for long read sequencing e.g. PacBio, Nanopore MinION. protocols.io https://dx.doi.org/10.17504/protocols.io.hadb2a6 Copy   

•   Source:

Authors: Jennifer Gin, Yan Chen, Christopher Petzold
Group: LBNL-omics
Summary: Recent improvements in the speed and sensitivity of liquid chromatography-mass spectrometry systems have driven progress toward system-wide characterization of the proteome of many species. These efforts create large proteomic datasets that provide insight into biological processes and identify diagnostic proteins whose abundance changes significantly under different experimental conditions. Consequently, it is important to have reproducible sample preparation methods that consist of mixing, various centrifugation and incubation steps, and an extended tryptic digestion step. We developed a high-throughput sample preparation workflow that consists of cell lysis, protein precipitation, protein resuspension, protein quantification, and normalization of protein concentration followed by standard bottom-up proteomic procedures of reducing and blocking cysteine residues and tryptic digestion.This protocol was adapted from the manual sample preparation method found in Chen, Y., et al. "Automated “Cells-To-Peptides” Sample Preparation Workflow for High-Throughput, Quantitative Proteomic Assays of Microbes."Journal of proteome research 18.10 (2019): 3752-3761.

Proper citation: Jennifer Gin, Yan Chen, Christopher Petzold 2020. Chloroform-Methanol Protein Extraction for Gram-negative Bacteria (High Throughput). protocols.io https://dx.doi.org/10.17504/protocols.io.bfx6jpre Copy   

•   Source:

Authors: Daniel Marchal
Summary: This cloning protocol refers to the Marburg Collection

Proper citation: Daniel Marchal 2018. Golden Gate Cloning LVL 2. protocols.io https://dx.doi.org/10.17504/protocols.io.uview4e Copy   

•   Source:

Authors: Alan Cone
Group: Ju Lab
Summary: Fresh Protein Extraction Sample Buffer.

Proper citation: Alan Cone 2015. Yeast Protein Extraction Sample Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.ebmbak6 Copy   

•   Source:

Authors: Vasily Pavelko

Proper citation: Vasily Pavelko 2021. Recan: R-based tool for detection of recombination in viral genomes. protocols.io https:// Copy   

•   Source:

Authors: Grieg F. Steward and Alexander I. Culley
Group: VERVE Net
Summary: This protocol is a minor modification of that reported by Culley and Steward (2007). As the starting point for this protocol, we assume that viruses have been collected on an aluminum oxide 0.02-µm syringe-tip filter (Anotop, Whatman), but other filters capable of capturing viruses may be substituted.This is a protocol from: Steward, G. F. and A. I. Culley. 2010. Chapter 16: Extraction and purification of nucleic acids from viruses. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7Please see the published manuscript for additional information.

Proper citation: Grieg F. Steward and Alexander I. Culley 2015. Extracting nucleic acids from viruses on a filter. protocols.io https://dx.doi.org/10.17504/protocols.io.dsm6c5 Copy   

•   Source:

Authors: Angel Justiz-Vaillant, Belkis Ferrer-Cosme
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. [1]The immunoregulatory cytokine IL-41 (also known as meteorin-like protein) is expressed at high levels in the synovium of patients with psoriatic arthritis (PsA).[2]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.2.Onuora S. Novel cytokine, IL-41, linked with PsA.Nat Rev Rheumatol. 2019;15(11):636. doi:10.1038/s41584-019-0314-7

Proper citation: Angel Justiz-Vaillant, Belkis Ferrer-Cosme 2020. ELISA for quantification of Granulocyte-colony stimulator factor (G-CSF) in human serum or plasma. protocols.io https://dx.doi.org/10.17504/protocols.io.bkspkwdn Copy   

•   Source:

Authors: Mike Gibson
Summary: Endo F1 cleaves Asparagine-linked high mannose or hybrid oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact.

Proper citation: Mike Gibson 2018. Endo F1 Protocol for Deglycosylating Glycoproteins. protocols.io https://dx.doi.org/10.17504/protocols.io.sebeban Copy   

•   Source:

Authors: Aymara Mas, Alba Machado-Lopez, Patricia Escorcia, Carlos Simón
Summary: This protocol describes the procedure for dissociating myometrial samples, which is based in an enzymatic disaggregation using Collagenase IV and DNAse I. This protocol is adapted from Mas et al. 2012 Fertil Steril with minor modifications.

Proper citation: Aymara Mas, Alba Machado-Lopez, Patricia Escorcia, Carlos Simón 2020. Enzymatic disaggregation of human myometrium for 10X Single Cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bb5miq46 Copy   

•   Source:

Authors: Edward Lebrun
Summary: Protocol for conducting intraspecific aggression assays with the tawny crazy,Nylanderia fulva.

Proper citation: Edward Lebrun 2019. Intaspecific aggression assay for Nylanderia fulva. protocols.io https://dx.doi.org/10.17504/protocols.io.8z9hx96 Copy   

•   Source:

Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Rachele Cesaroni 2017. Euplotes crassus transfection through microinjection into the macronucleus. protocols.io https://dx.doi.org/10.17504/protocols.io.hi7b4hn Copy   

•   Source:

Authors: Hongwei Dong
Summary: Stereotaxic surgeries are performed to inject neural circuit tracers into different target regions in the brain.

Proper citation: Hongwei Dong 2019. Quadruple Retrograde Tracing. protocols.io https://dx.doi.org/10.17504/protocols.io.4xpgxmn Copy   

•   Source:

Authors: Tjusls China
Summary: High Throughput Screens (HTS) are recent scientific methods relevant to the field of chemistry and biology, in which hundreds of thousands of experimental samples are subjected to simultaneous testing under given conditions. The sample themselves may take the form of biochemical agents such as chemical compounds, amino acids, or live cells. With the development of laboratory robotics that automate sample preparation, handling and data analysis, scientists can easily and reliably generate and use large datasets from these HTS to answer complex biological questions. HTS is now widely used in the field of pharmaceuticals, biotech and academic institutes for drug discovery, target validation and the identification of genes or proteins that modulate a particular biological pathway.* Referencehttps://www.singerinstruments.com/resource/what-is-high-throughput-screening/

Proper citation: Tjusls China 2019. 16 High Throughput Screening with Fluorescent Probe. protocols.io https://dx.doi.org/10.17504/protocols.io.6guhbww Copy   

•   Source:

Authors: Amy Zimmerman
Group: VERVE Net, Worden Lab
Summary: Purpose: To reproducibly generate fresh preparations of virus for independent experiments.Fresh virus sample for an experiment is generated by a primary infection of exponentially growing host cells from a master stock of virus. The infected host is allowed to lyse until the culture is cleared. The lysate is filtered to remove any large cellular debris, then the viral-size fraction is concentrated from the filtered lysate and washed with buffer using a centrifugal concentrator.  The viral concentrate is stored at 4°C protected from light and should be used for an experiment within 1-2 days. On the day of (or the day before) the experiment, an MPN assay should be set up to assess infectivity of the fresh viral concentrate.

Proper citation: Amy Zimmerman 2017. Propagation of marine eukaryotic viruses (Prasinoviruses). protocols.io https://dx.doi.org/10.17504/protocols.io.gujbwun Copy   

•   Source:

Authors: Jeric Harper
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Alverson Lab

Proper citation: Jeric Harper 2016. Diatom Transformation via Bacterial Conjugation. protocols.io https://dx.doi.org/10.17504/protocols.io.fp7bmrn Copy   

•   Source:

Authors: Adria D. Lee, Pamela K. Cassiday, Lucia C. Pawloski, Kathleen M. Tatti, Monte D. Martin, Elizabeth C. Briere, M. Lucia Tondella, Stacey W. Martin

Proper citation: Adria D. Lee, Pamela K. Cassiday, Lucia C. Pawloski, Kathleen M. Tatti, Monte D. Martin, Elizabeth C. Briere, M. Lucia Tondella, Stacey W. Martin 2020. Standard Operating Procedure for Real-time PCR Detection and Identification of Bordetella pertussis, B. parapertussis, and B. holmesii using AB7500. protocols.io https:// Copy   

•   Source:

Authors: Curtis Huttenhower
Group: VERVE Net, Huttenhower Lab
Summary: In this protocol we describe some approaches to graphically represent single profiled samples or a merged table of relative abundances.

Proper citation: Curtis Huttenhower 2016. GraPhlAn visualization of single and multiple samples. protocols.io https://dx.doi.org/10.17504/protocols.io.d9s96d Copy   

•   Source:

Authors: Mark Moosburner, Andrew Allen
Group: A.E. Allen Lab
Summary: Bacterial-conjugation methodology used to introduce the CRISPR-Cas9 episome generated using the GG2 assembly protocol.This method, though, can be adapted for any episomal transfromation from an E. coli culture that contains a cargo plasmid (episome) and conjugation plasmid (pta-MOB) to Phaeodactylum.

Proper citation: Mark Moosburner, Andrew Allen 2019. CRISPR-Cas9 episome conjugation into Phaeodactylum tricornutum. protocols.io https://dx.doi.org/10.17504/protocols.io.5pvg5n6 Copy   

•   Source:

Authors: Guillermo Fernández Rodríguez
Group: AEGIS - Madrid iGEM 2019
Summary: ELONA (Enzyme-Linked Oligonucleotide Assay), is a biochemical method based on enzyme linked immunosorbent assay (ELISA) that allows to demonstrate that aptamers selected by SELEX can be effective and useful as biorecognition molecules and laboratory tools. The ELONA format choosed uses an anti-digoxigenin antibody to recognize an aptamer previously labelled with digoxigenin. This antibody is conjugated with a peroxidase enzyme, and once it adds ABTS solution, it will be responsible for the colourimetric reaction which will be detected. (We used 5 replicates per dilution)

Proper citation: Guillermo Fernández Rodríguez 2019. Cell-ELONA. protocols.io https://dx.doi.org/10.17504/protocols.io.7umhnu6 Copy   

•   Source:

Authors: Smruthi Karthikeyan, Greg Humphrey
Group: Coronavirus Method Development Community
Summary: Large-scale wastewater surveillance has the ability to greatly augment the tracking of infection dynamics especially in communities where the prevalence rates far exceed the testing capacity. However, current methods for viral detection in wastewater are severely lacking in terms of scaling up for high throughput. In the present study, we employed an automated magnetic-bead based concentration approach for viral detection in sewage that can effectively be scaled up for processing 24 samples in a single 40-minute run. The method compared favorably to conventionally used methods for viral wastewater concentrations with a limit of detection of 8.809 viral gene copies/ml from input sample volumes as low as 10ml and can enable the processing of over 100 wastewater samples in a day.

Proper citation: Smruthi Karthikeyan, Greg Humphrey 2020. Automated high throughput viral concentration from wastewater using the KingFisher Flex platform. protocols.io https://dx.doi.org/10.17504/protocols.io.bptemnje Copy   

•   Source: