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Authors: Judy Northill
Summary: This RT-PCR will detected Aichivirus A from human samples. It spans the junction region of 3C and 3D and is used for genotyping.
Proper citation: Judy Northill 2019. Aichivirus 3C3D RT-PCR. protocols.io dx.doi.org/10.17504/protocols.io.2i7gchn Copy
Authors: Devin Willis
Group: XPRIZE Rapid Covid Testing, RNAPath
Proper citation: Devin Willis 2020. RNAPath Target Identification via Fluorescent Hybridization. protocols.io dx.doi.org/10.17504/protocols.io.bk37kyrn Copy
Authors: Andreea S
Group: iGEM Groningen 2020
Summary: The potato root secretes root exudates, a mixture of organic molecules out of which solanine is found in the highest concentration (range of ug/ml). Also, solanine is a molecule specifically found in the root exudate of potato plants, which makes it a good candidate for a dependecy molecule which will ensure that Bacillus mycoides doesn't escape the designated action area. Studies have shown that there are soil bacteria able to metabolize solanine as a carbon source. Unfortunately, we weren't able to find an already described solanine-inducing promoter. Because the natural habitat of B. mycoides is the potato rhizosphere, we have strong reasons to believe that if such a promoter may already be present in its genome. In order to find this promoter, we designed a CHIP-Seq experiment. B. mycoides will be incubated with a different range of solanine concentrations. The DNA will be extracted and the CHIP-Seq technique will be used to detect potential binding sites of solanine operators using monoclonal anti-solanine antibodies.
Proper citation: Andreea S 2020. Transcriptomics. protocols.io dx.doi.org/10.17504/protocols.io.bme9k3h6 Copy
Authors: DAVID DUNIGAN AND IRINA AGARKOVA
Group: VERVE Net
Summary: For use in Polyacrylamide Gel System For Electrophoresis Of Proteins.
Proper citation: DAVID DUNIGAN AND IRINA AGARKOVA 2016. Resolving gel buffer (4X). protocols.io dx.doi.org/10.17504/protocols.io.eq3bdyn Copy
Authors: Sarah Giuliani
Summary: Experiment purpose is to monitor the time-course of a large-scale infection of host cyanobacteria by phage under variable media conditions and obtain samples for proteomic and transcriptomic analysis.As a minimum, prepare 12 sample bottles for cells infected with phage and control phage lysate, under 2 different media treatments, with triplcates of each unique Phage/Treatment combination.
Proper citation: Sarah Giuliani 2016. Setting up Experimental Cultures for Large-scale One-step Phage Infection of Cyanobacteria. protocols.io dx.doi.org/10.17504/protocols.io.fktbkwn Copy
Authors: Nick Irwin, Elisabeth Hehenberger, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Caecitellus sp. is a small phagotroph belonging to the biocosoecid order of the stramenopiles. Caecitellus is found across global oceans but it's role in marine ecosystems, as with many heterotrophic flagellates, is unclear. We isolated a strain of Caecitellus (small subunit rRNA gene was 99.53% identical to Caecitellus parvulus and Caecitellus paraparvulus) from a contaminated culture of Oxyrrhis marina and assessed its amenability to transformation. Fluorescein isothiocyanate conjugated to dextran was successfully introduced into Caecitellus sp. using a high voltage exponential decay pulse (1000 V, 10 µF, ∞ Ω) from a BioRad GenePulser Xcell (cuvette width 0.2 cm). Cell viability was confirmed by observing motility and fluorescence 24 h after electroporation (Figure 1). Lower voltage pulses did not result in FITC uptake. Estimates of transformation effifiency were difficult to obtain due to the small size of the cells.Unfortunately, the isolated strain of Caecitellus perished in an incubator malfunction. However, these results may be transferable to Caecitellus parvulus, a model for heterotrophic flagellates.
Proper citation: Nick Irwin, Elisabeth Hehenberger, Patrick Keeling 2019. Electroporation of Caecitellus sp. with FITC-dextran. protocols.io dx.doi.org/10.17504/protocols.io.35kgq4w Copy
Authors: Center for Epigenomics UCSD
Group: LungMap2 Consortium
Summary: Protocol describing nuclei isolation from frozen tissue including lung using grinding for droplet-based single nucleus RNA-seq.
Proper citation: Center for Epigenomics UCSD 2020. Nuclei preparation from human lung using grinding for snRNA-seq. protocols.io dx.doi.org/10.17504/protocols.io.bprdmm26 Copy
Authors: Ajit N Shah
Proper citation: Ajit N Shah 2019. AccuBlue®Broad Range RNA Quantitation. protocols.io dx.doi.org/10.17504/protocols.io.87bhzin Copy
Authors: Rob Parks, Barton Haynes
Group: XPRIZE Rapid Covid Testing
Proper citation: Rob Parks, Barton Haynes 2020. SARS-CoV-2 Antigen Detection ELISA. protocols.io dx.doi.org/10.17504/protocols.io.bk2ckyaw Copy
Authors: Jian Gong
Group: Bosak Lab
Summary: This protocol describes the adaption of a silica colorimetric assay originally described in Strickland and Parsons, 1972 (p65-70), modified for use on a multi-mode plate reader spectrophotometer (BioTek, Synergy 2, Winooski, VT, USA), using standard 96-well plates for rapid measurements of 1 mL, diluted water samples. This assay measures the concentration of molybdate-reactive silica (monomeric and short polymers of silicic acid) in solution.Samples for this assay should be filtered (0.2 µm syringe filter) and placed in 2 mL microcentrifuge tubes. Avoid sample freezing. Store samples in the dark whenever possible.
Proper citation: Jian Gong 2020. Dissolved silica colorimetric assay using a plate reader (96-well plate). protocols.io dx.doi.org/10.17504/protocols.io.bd7ni9me Copy
Authors: Oscar Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium
Group: Coronavirus Method Development Community
Summary: The following protocol describes the "4S" (Sewage, Salt, Silica and SARS-CoV-2) workflow applied to using dry silica powder as an RNA-binding matrix instead of silica spin columns. This offers an even more economical alternative, requiring only centrifugation to extract RNA from wastewater. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.
Proper citation: Oscar Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium 2020. Direct wastewater RNA extraction via the "Milk of Silica (MoS)" method - A companion method to "Sewage, Salt, Silica and SARS-CoV-2 (4S)". protocols.io dx.doi.org/10.17504/protocols.io.biwfkfbn Copy
Authors: Chin Yee Tan
Proper citation: Chin Yee Tan 2019. RNA isolation for tissue. protocols.io dx.doi.org/10.17504/protocols.io.zvqf65w Copy
Authors: Carol Stepien, Anna Elz, Matthew Snyder
Summary: This protocol describes the data collection and analysis performed in "Invasion genetics of the silver carp (Hypophthalmichthys molitrix) across North America: Differentiation of fronts, introgression, and eDNA detection". Published in PLOS One, 2018.The invasive silver carp Hypophthalmichthys molitrix escaped from southern U.S. aquaculture during the 1970s to spread throughout the Mississippi River basin and steadily moved northward, now reaching the threshold of the Laurentian Great Lakes. The silver carp is native to eastern Asia and is a large, prolific filter-feeder that decreases food availability for fisheries. The present study evaluates its population genetic variability and differentiation across the introduced range using 10 nuclear DNA microsatellite loci, sequences of two mitochondrial genes (cytochrome b and cytochrome c oxidase subunit 1), and a nuclear gene (ribosomal protein S7 gene intron 1). Populations are analyzed from two invasion fronts threatening the Great Lakes (the Illinois River outside Lake Michigan and the Wabash River, leading into the Maumee River and western Lake Erie), established areas in the southern and central Mississippi River, and a later Missouri River colonization. Results discern considerable genetic diversity and some significant population differentiation, with greater mtDNA haplotype diversity and unique microsatellite alleles characterizing the southern populations. Invasion fronts significantly differ, diverging from the southern Mississippi River population. About 3% of individuals contain a unique and very divergent mtDNA haplotype (primarily the southerly populations and the Wabash River), which may stem from historic introgression in Asia with female largescale silver carp H. harmandi. Nuclear microsatellites and S7 sequences of the introgressed individuals do not significantly differ from silver carp. MtDNA variation is used in a high-throughput sequence assay that identifies and distinguishes invasive carp species and their population haplotypes (including H. molitrix and H. harmandi) at all life stages, in application to environmental (e)DNA water and plankton samples. We discerned silver and bighead carp eDNA from 10 bait shops in the Great Lakes watershed, indicating that release from retailers comprises another likely vector. Our findings provide key baseline population genetic data for understanding and tracing the invasion’s progression, facilitating detection, and evaluating future trajectory and adaptive success.
Proper citation: Carol Stepien, Anna Elz, Matthew Snyder 2018. Protocols from: Invasion genetics of the silver carp (Hypophthalmichthys molitrix) across North America: Differentiation of fronts, introgression, and eDNA detection. protocols.io dx.doi.org/10.17504/protocols.io.u6zezf6 Copy
Authors: elisa.granato
Group: Elisa Granato, AG Wittbrodt
Proper citation: elisa.granato 2020. Quick-n-Dirty electrocompetent E. coli cells. protocols.io dx.doi.org/10.17504/protocols.io.bjqfkmtn Copy
Authors: Frank Aylward
Proper citation: Frank Aylward 2018. SYSB 3036 W01: Introduction to Unix. protocols.io dx.doi.org/10.17504/protocols.io.vqpe5vn Copy
Authors: Julia Moraes, Diego Bonatto
Group: Grupo de Estudos de Leveduras Cervejeiras
Summary: The growing of microbrewerys in Brazil has imposing an increase in the production of different supplies for brewing, iincluding yeast biomass for beer fermentation. In Brazil,only four companies produce and sell yeasts for microbreweries, and only two companies are able to attend microbreweries. Additionaly, these companies should be able to deliver yeast cells to different states and cities in Brazil. During transport, the yeast cells are exposed to thermical stress (due to the lack of temperature control) and chronological aging (due to the long period of storage and transport). When delivered to the microbreweries, those yeast cells display problems during beer fermentation as the consequence of a loweryeast vitality and viability and affecting beer’s quality. Thus, in this work a new colorimetric assay was developed to access the vitality of yeast cells by using resazurin. Resazurin is a highly dichromatic dye, showing a colorimetric change proportional to the yeast cellvitality that can be easily visualized by naked eye. To evalute the potential of resazurin to differentiate yeast cells with low and high vitality, different yeast ale and lager strains (US-05 and W34/70, respectively) were submitted to heat shock stress and their vitality was accessed by resazurin, testing different variables as concentration of work solution, cellular density and incubation time. The resazurin data, supported by acidification power test, showed a vitality decrese in cells submited to heat shock stress. Futhermore, the sensibility of the resazurin assay was explored during chronological aging of yeast cells. The assay was capable to detect differences of vitality and viability between yeast strains. In addition, the potencial of the resazurin test to predict the fermentative capacity of stressed and non-estressed yeast cells was observed in wort attenuation assays, comfirming the aplicability of resazurin in the microbrewery context.
Proper citation: Julia Moraes, Diego Bonatto 2018. Development of resazurin assay for evaluation of yeast viability and vitality in microbreweries (in Portuguese). protocols.io dx.doi.org/10.17504/protocols.io.pnedmbe Copy
Authors: Marconi Augusto Aguiar dos Reis, Roberta Maia C Romanelli, Zilma Reis
Summary: The authors detailed describe the standard procedures for immaturity-related respiratory disorders diagnosis during the first 72h of life. These disorders are respiratory distress syndrome of newborn (RDS) and transient tachypnea of the newborn (TTN), secondary outcomes in the International Clinical Trials Registry Platform under the numbers RBR-3f5bm5 and RBR33rnjf. It shall be used by the multicenter team of researchers, duly trained following the Good Clinical Practice Protocol, during the prospective evaluation of enrolled newborns to RDS and TTN diagnosis. Also, this protocol is corresponding documentation for the scientific publications related to the clinical trials: "Prematurity detection evaluating interaction between the skin of the newborn and light: protocol for the preemie-test multicentre clinical trial in Brazilian hospitals to validate a new medical device." "Premature or small for gestational age? International multicenter trial protocol for classification of the low birth weight newborn through the optical properties of the skin."
New version on 01/26/2020 to adjust typos in steps subheadings
Proper citation: Marconi Augusto Aguiar dos Reis, Roberta Maia C Romanelli, Zilma Reis 2020. Respiratory distress syndrome of the newborn and transient tachypnea of the newborn diagnosis . protocols.io dx.doi.org/10.17504/protocols.io.bbsninde Copy
Authors: James Angstman
Group: The Center for Genome Editing and Recording
Summary: RoboPreps are great for high-throughput cloning and subcloning, especially if you’re going to be doing Maxipreps afterward anyway. RoboPreps must be done in 96 well format as of now, though future generations may one day break through this technological barrier.
Proper citation: James Angstman 2019. BiomekFXp Robot Minipreps (RoboPreps). protocols.io dx.doi.org/10.17504/protocols.io.3zqgp5w Copy
Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Proper citation: Matthew Sullivan 2016. DNA Precipitation Protocol. protocols.io dx.doi.org/10.17504/protocols.io.c34yqv Copy
Authors: Veronika Cencen
Proper citation: Veronika Cencen 2019. Unroofing mammalian cells for AFM. protocols.io dx.doi.org/10.17504/protocols.io.z2zf8f6 Copy
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