Protocols   Select Another Resource Report Type

Authors: Linbin Huang, Xinghong Yan
Summary: Pyropia yezoensis is one of the most valuable and widely cultivated seaweeds across the world. However, there is limited data regarding its genetic background underlying complex economic traits. The molecular genetic linkage map of P. yezoensis was not constructed. The most probable cause was the lack of mapping population, which was the basic of linkage analysis. In the present study, a red-type and fast-growing pigmentation mutant Py-HT and a wild-type strain Py-LS who had enough variation for traits of interest at both DNA and phenotypic level were crossed under control. The heterozygote (heterozygous conchocelis) was identified among conchocelis colonies which were developed from single zygotospore released from zygotosporangia of fertilized Py-HT blade, based on the result that 91.9% F1 gametophytic blades developed from the conchospores of heterozygous conchocelis were linearly sectored with 2-4 color sectors of parental colors. Afterwards, 57 chimeric blades with four color sectors were screened from thousands of F1 blades and 228 color sectors were separated according the boundaries of color sectors for single culture. A single zygotospore released from zygotosporangia of one sector through selfing was selected and then developed into homozygous conchocelis, which was declared a double haploid strain. Finally, a mapping population with 148 strains obtained from 37 of the 57 four color sectored blades was developed, which was used for the construction of genetic linkage maps and analysis of quantitative trait loci of P. yezoensis blades.

Proper citation: Linbin Huang, Xinghong Yan 2019. Development of a doubled haploid mapping population of Pyropia yezoensis and measurement of the economic characters of blade. protocols.io https://dx.doi.org/10.17504/protocols.io.x3vfqn6 Copy   

•   Source:

Authors: Matema Imakumbili
Summary: This protocol describes how to collect and prepare a soil sample for various soil chemical analyses.

Proper citation: Matema Imakumbili 2019. Soil Sampling and Preparation for Soil Chemical Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.x72frqe Copy   

•   Source:

Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Hydrogen peroxide (H2O2) is a ubiquitous, toxic, metabolic by-product of aerobic respiration, oxidative stress, and oxidative injury. Cayman's Hydrogen Peroxide Assay Kit utilizes the well established xylenol orange detection method of quantifying the oxidation of ferrous ions (Fe2+) to ferric ions (Fe3+) by hydrogen peroxide. A unique feature of Cayman's assay is the inclusion of catalase as an H2O2 scavenger for the purpose of confirming the specificity of the reaction for H2O2. The sensitivity and the specificity of the assay make it well suited to accurately measure urinary levels H2O2 in a 96 well plate format. Each kit contains hydrogen peroxide, reagent 1, reagent 2, catalase, a 96 well plate, plate cover, and complete instructions.

Proper citation: Peter Havel 2019. UC Davis - Hydrogen Peroxide. protocols.io https://dx.doi.org/10.17504/protocols.io.ypqfvmw Copy   

•   Source:

Authors: Josh Quick, Kirstyn Brunker
Summary: This is a ‘one-pot ligation’ protocol for Oxford Nanopore native barcoded ligation libraries using shearing.

Proper citation: Josh Quick, Kirstyn Brunker 2020. One-pot native barcoding of amplicons (Ultra II AMII ligation). protocols.io https://dx.doi.org/10.17504/protocols.io.bdaqi2dw Copy   

•   Source:

Authors: Bonnie Hurwitz, Ken Youens-Clark
Group: VERVE Net, Hurwitz Lab
Summary: K-mer-based approaches to determining sequence similiarity can be faster than traditional use of BLAST.  This application uses Jellyfish to index host FASTA files, then uses the mode value (default 2) of the number of matching k-mers (default k=20) from the input sequence to determine whether the sequence is similar enough to the host sequence to be rejected.  The output of the app is:"screened" directory containing the sequences from each file that were found to be dissimlar to the host"rejected" directory containing the sequence from each file that were too similar to the host"jf" directory containing Jellyfish indexes of each "host" file (useful for later runs with other files to skip recreating)"kmer" directory containing k-mers of query sequences and ".loc" file showing the number of ".kmer" lines associated to each sequence Code is freely available at Github.

Proper citation: Bonnie Hurwitz, Ken Youens-Clark 2016. K-mer-based host screening. protocols.io https://dx.doi.org/10.17504/protocols.io.ehjbb4n Copy   

•   Source:

Authors: Amy Lyden, Gloria Castañeda, Emily Crawford, Saharai Caldera, Jenai Quan, Katherine Travisano
Group: Chan Zuckerberg Biohub
Summary: FLASH is a crispr-cas9 technology that enriches for targeted sequences in sequencing libraries. The initial DNA sample undergoes a blocking step that removes of the 5’ phosphoryl groups of the DNA fragments, resulting in a product that is not amendable for downstream adaptor ligation or amplification via standard Illumina-based library preparation. The subsequent incorporation of targeted CRISPR-cas9 library exposes the desired regions of interest, allowing them to be processed into a library. For more information on methods and results, please see the FLASH paper. For FLASH guide RNA design help, please see our github.

Proper citation: Amy Lyden, Gloria Castañeda, Emily Crawford, Saharai Caldera, Jenai Quan, Katherine Travisano 2019. FLASH v2.0. protocols.io https://dx.doi.org/10.17504/protocols.io.6iwhcfe Copy   

•   Source:

Authors: Bao Thai
Group: Stephen Floor Lab

Proper citation: Bao Thai 2018. Cas9/sgRNA ribonucleoprotein nucleofection using Lonza 4D nucleofector. protocols.io https://dx.doi.org/10.17504/protocols.io.vnce5aw Copy   

•   Source:

Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the preparation of 1x3 glass slides for mounting embedded mouse brain samples for subsequent imaging and sectioning on TissueCyte 1000. Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Proper citation: Allen Institute for Brain Science 2020. Slide Preparation for Mounting Samples for TissueCyte 1000 Sectioning and Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.bdvni65e Copy   

•   Source:

Authors: Marzia Ognibene

Proper citation: Marzia Ognibene 2020. Western blot analysis and immunoprecipitation assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bgecjtaw Copy   

•   Source:

Authors: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda
Group: Coronavirus Method Development Community
Summary: This protocol is folked from "ARTIC amplicon sequencing protocol for MinION for nCoV-2019" by Josh Quick to adapt it to illumina sequencers. In this Version V3, the reverse-transcription step was changed to use of NEB's LunaScript RT SuperMix Kit instead of Thermo Fisher's SuperScript IV Reverse Transcriptase as adapting to the recent update of ARTIC Network's protocol (LoCost). Tyson et al., Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore bioRxiv2020.09.04.283077;doi:https://doi.org/10.1101/2020.09.04.283077 Because the PCR products are fragmented before ligated with adapters, this protocol is compatible to many sequencing kits with various read lengths (75PE, 150PE, etc.) in Illumina machines. While the library preparation uses QiaSeq FX by Qiagen and is basically straight forward (as par kit instruction but modified to 1/4 scale), some tweaks for much of simplicity and speed were added.Change histories (V2): The amount of cDNA input to multiplex PCR is increased (now almost same amount to the ARTIC Network's original protocol) (Step 10). This change gives better results for samples with extremely low RNA copy. Amount of adapter solution input was corrected (Step 19).Change histories (V3): RT step has been changed to use of LunaScript (Tyson et al., 2020).Corrected typos and wrong descriptions about amount of reagents.Added a link to library quantification protocol.Other minor changes.Change histories (V3): RT step has been changed to use of LunaScript (Tyson et al., 2020).Corrected typos and wrong descriptions about amount of reagents.Added a link to library quantification protocol.Other minor changes.

Proper citation: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda 2020. nCoV-2019 sequencing protocol for illumina. protocols.io https://dx.doi.org/10.17504/protocols.io.bnn7mdhn Copy   

•   Source:

Authors: Laura Ruiz Remolina

Proper citation: Laura Ruiz Remolina 2017. Western blot. protocols.io https://dx.doi.org/10.17504/protocols.io.j8bcrsn Copy   

•   Source:

Authors: George more

Proper citation: George more 2020. Bitdefender  does not update automatically 2020. protocols.io https:// Copy   

•   Source:

Authors: Sam Mugford, Roland Wouters, Thomas C Mathers, Saskia Hogenhout
Summary: Genomic studies of natural populations frequently benefit by having data from single individuals. This approach has a number of advantages: it avoids the risk of accidently pooling together individuals of closely related species, and it enables better resolution of haplotypes by separating non-identical individuals. However, especially in the case of very small invertebrates, recovering sufficient nucleic acid from individual samples can be challenging. Here we present a method suitable for extraction of DNA from single individual aphids (weighing approximately 0.5mg). We are able to recover 100’s of ng of high-quality DNA suitable for whole-genome sequencing using short-read technologies. DNA extraction using Cetrimonium bromide (CTAB) is a tried and tested method, and the protocol presented here is adapted from that of Marzachì et al. (1998). We present some modifications to optimize this popular method to maximize yield and quality from small single insect samples.We have used this method across a range of aphid species, and also other sap-sucking hemipteran insects. It has enabled sequencing and de-novo assembly of a range of species from single individuals collected from the field, and also resequencing of individuals of the model aphid Myzus persicae. This is compatible with the method of ambient storage of dehydrated filed collected samples we describe in Wouters et al. (2020).Marzachì, C, Veratti, F, & Bosco, D (1998). Direct PCR detection of phytoplasmas in experimentally infected insects. Annals of Applied Biology, 133(1), 45-54.Wouters, R; Mugford S, & Hogenhout, S (2020). Ambient sample storage system of field-collected insect samples for genomics. protocols.io dx.doi.org/10.17504/protocols.io.bgxnjxme

Proper citation: Sam Mugford, Roland Wouters, Thomas C Mathers, Saskia Hogenhout 2020. High quality DNA extraction from very small individual insects. protocols.io https://dx.doi.org/10.17504/protocols.io.bg6wjzfe Copy   

•   Source:

Authors: Ziqiang Chen
Group: GigaScience Press, BGI
Summary: BGISEQ-500 is a desktop sequencer developed by BGI. This protocol adjusts the process in order to apply the 10X contruction to the BGISEQ-500 rather than illumina.

Proper citation: Ziqiang Chen 2019. BGISEQ-500 10X library construction. protocols.io https://dx.doi.org/10.17504/protocols.io.3jagkie Copy   

•   Source:

Authors: Morrisey Lab
Group: LungMap2 Consortium

Proper citation: Morrisey Lab 2021. Human Lung Digestion: Deriving a single cell suspension. protocols.io https:// Copy   

•   Source:

Authors: Chris Berndsen

Proper citation: Chris Berndsen 2021. Determination of the accuracy of micropipettes for the biochemistry lab. protocols.io https:// Copy   

•   Source:

Authors: New England Biolabs, Ken Christensen
Summary: The following is a "typical" restriction endonuclease reaction. Please see the "guidelines" tab below for the NEB tips on optimizing restriction digests.

Proper citation: New England Biolabs, Ken Christensen 2020. Restriction Digest -- CHEM 584. protocols.io https://dx.doi.org/10.17504/protocols.io.bk5yky7w Copy   

•   Source:

Authors: Ana Cristina Jaramillo Madrid, Justin Ashworth
Summary: This protocol has been successfully used to express nourseothricin resistance gene, mVenus fluorescence protein and other proteins related to triterpenoids production in Thalassiosira pseudonana (Tp) strain CCMP1335. The original protocol was published by Karas et al. (2015) where a detailed description of L1 medium and plates preparation is presented.

Proper citation: Ana Cristina Jaramillo Madrid, Justin Ashworth 2019. Tranformation of Thalassiosira pseudonana via bacterial conjugation. protocols.io https://dx.doi.org/10.17504/protocols.io.7fghjjw Copy   

•   Source:

Authors: Sam Li
Group: BioLegend
Summary: Precision Count Beads™ are designed for counting the absolute number of cells in a complex mix population and other particles by flow cytometry. Precision Count Beads™ are excited by a variety of lasers including violet (405nm), blue (488nm), yellow/green (562nm), and red (633nm).

Proper citation: Sam Li 2019. Precision Count Beads™ Protocol and Applications. protocols.io https://dx.doi.org/10.17504/protocols.io.baariad6 Copy   

•   Source:

Authors: ayten
Group: XPRIZE Rapid Covid Testing

Proper citation: ayten 2020. Easy Covid LAMP. protocols.io https://dx.doi.org/10.17504/protocols.io.bk6gkzbw Copy   

•   Source: