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Authors: New England Biolabs
Group: New England Biolabs (NEB)
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 2014. Restriction Digest. protocols.io dx.doi.org/10.17504/protocols.io.chkt4v Copy
Authors: Afiahayati, Sato K, Namiki T, Hachiya T, Tanaka H, Sakakibara Y.
Group: VERVE Net, Hurwitz Lab
Summary: Motivation: An important step of "metagenomics" analysis is the assembly of multiple genomes from mixed sequence reads of multiple species in a microbial community. Most conventional pipelines employ a single-genome assembler with carefully optimized parameters and post-process the resulting scaffolds to correct assembly errors. Limitations of the use of a single-genome assembler for de novo metagenome assembly are that highly conserved sequences shared between different species often causes chimera contigs, and sequences of highly abundant species are likely mis-identified as repeats in a single genome.Methods:We modified and extended a single-genome and de Bruijn-graph based assembler, Velvet, for de novo metagenome assembly. Our fundamental ideas are first decomposing de Bruijn graph constructed from mixed short reads into individual sub-graphs and second building scaffolds based on every decomposed de Bruijn sub-graph as isolate species genome.
Proper citation: Afiahayati, Sato K, Namiki T, Hachiya T, Tanaka H, Sakakibara Y. 2016. Installation and Getting Started. protocols.io dx.doi.org/10.17504/protocols.io.d5k84v Copy
Authors: Kelsey Knight
Group: BioLegend
Proper citation: Kelsey Knight 2018. Immunohistochemistry Protocol for Paraffin-Embedded Sections. protocols.io dx.doi.org/10.17504/protocols.io.tkjekun Copy
Authors: Alexandra Ehl, David Frommholz, Nadine Stefanczyk
Summary: Purification Guide for the Isolation of Antibodies with ChroPlate Filtration Plates by DALEX Biotech.Easy and quick high throughput antibody purification from various sources and species.Each well of the ChroPlate has a binding capacity of > 1 mg (tested with human polyclonal Ig, binding varies between species and clones).The proprietary resin does not shrink or swell in aqueous buffers.High pressure stability.pH stability short term 2 - 8, long term 3 - 8.Excellent thermal stability up to 15 minutes at 80 °C in aqueous buffers at neutral pH.Can be dried for long term storage (80 °C for > 2 h).
Proper citation: Alexandra Ehl, David Frommholz, Nadine Stefanczyk 2018. ChroPlate - ProteinA. protocols.io dx.doi.org/10.17504/protocols.io.uycexsw Copy
Authors: Dr.maria Yousuf
Proper citation: Dr.maria Yousuf 2018. Bioinformatics: A rational combine approach used for the identification and in-vitro activity evaluation of potent β-Glucuronidase inhibitors. protocols.io dx.doi.org/10.17504/protocols.io.q6pdzdn Copy
Authors: Brooks Lab University of California, Eva Robinson, Alison Tang
Group: BrooksLabUCSC
Summary: This is a Western Blotting Protocol for Brooks Lab, Department of Biomolecular Engineering, University of California, Santa Cruz.
Proper citation: Brooks Lab University of California, Eva Robinson, Alison Tang 2020. Brooks Lab Western Blotting Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bcsmiwc6 Copy
Authors: Neilier Junior
Summary: This protocol has been standardized for analysis of protease inhibitors in soybean leaf extract, but can be easily adjusted for other biological samples.
Proper citation: Neilier Junior 2019. Enzymatic Assay of Trypsin Inhibition. protocols.io dx.doi.org/10.17504/protocols.io.9a4h2gw Copy
Authors: Sarah Giuliani
Group: Coleman Lab
Summary: 15 Hourly Timepoints: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14Sampling is for qPCR assay to determine total extracellular phage (qEXT) and MPN (most probable number) assay to determine % phage infectivity.For qEXT and MPN sampling, 250 µL of sample in duplicates were collected and filtered through a 0.2 µm, 96-well filter plate, for each time point. (Flow through filtrate containing phage is the sample to analyze).
Proper citation: Sarah Giuliani 2016. Sampling for qEXT and MPN assays: Large-scale One-step Phage Infection of Cyanobacteria. protocols.io dx.doi.org/10.17504/protocols.io.fh3bj8n Copy
Authors: Yuanjun Yang, Fang Liu
Summary: Potato tubers (250 g) were washed with distilled water and smashed in 500 mL of a mixture containing 50% alcohol and 1.6% citric acid. The resulting mixture was filtrated twice and centrifuged at 4000 rpm. The supernatant was transferred into a column filled with the pretreated macroporous resin AB-8 for 6 h, and the pigments absorbed in AB-8 were eluted with 95% alcohol. The eluate was concentrated with rotary evaporator at 30 °C and dried in vacuo for 12 h. Two biological replicates were used in this study.
Proper citation: Yuanjun Yang, Fang Liu 2017. potato anthocyanin extraction and purification. protocols.io dx.doi.org/10.17504/protocols.io.k4mcyu6 Copy
Authors: Andrew Loudon
Group: ParfreyLab
Proper citation: Andrew Loudon 2016. Sea stars bacterial sampling. protocols.io dx.doi.org/10.17504/protocols.io.f7abrie Copy
Authors: Priota Islam
Group: Behavioural Genomics
Summary: C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar which has been aseptically poured into petri plates. Smaller plates (35 mm diameter) are useful for mating or when using expensive drugs. Medium size plates (60 mm diameter) are useful for general strain maintenance, and larger plates (100 mm diameter) are useful for growing larger quantities of worms, such as for certain mutant screens. The NGM agar medium can be poured into petri plates easily and aseptically using a peristaltic pump. This pump can be adjusted so that a constant amount of NGM agar is dispensed into each petri plate. A constant amount of agar in the plates reduces the need for refocusing the microscope when you switch from one plate to another. Reference: http://www.wormbook.org/
Proper citation: Priota Islam 2019. Plate Pouring. protocols.io dx.doi.org/10.17504/protocols.io.3yygpxw Copy
Authors: Geoffrey W. Meissner, Jonathan B. Grimm, Rebecca M. Johnston, Ben Sutcliffe, Julian Ng, Gregory S.X.E. Jefferis, Sebastian Cachero, Luke D. Lavis, Oz Malkesman
Summary: The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.
Proper citation: Geoffrey W. Meissner, Jonathan B. Grimm, Rebecca M. Johnston, Ben Sutcliffe, Julian Ng, Gregory S.X.E. Jefferis, Sebastian Cachero, Luke D. Lavis, Oz Malkesman 2018. Polarity Hybrid Chemical Tag & IHC for Adult CNS. protocols.io dx.doi.org/10.17504/protocols.io.nycdfsw Copy
Authors: Didier Barradas-Bautista, Matias Alvarado, Mark Agostino, Germinal Cocho
Summary: This work aims for modeling and simulating the metastasis of cancer, via the analogy between the cancer process and the board game Go. In the game of Go, black stones that play first could correspond to a metaphor of the birth, growth, and metastasis of cancer. Moreover, playing white stones on the second turn could correspond the inhibition of cancer invasion. Mathematical modeling and algorithmic simulation of Go may, therefore, benefit the efforts to deploy therapies to surpass cancer illness by providing insight into the cellular growth and expansion over a tissue area. We use the Ising Hamiltonian, that models the energy exchange in interacting particles, for modeling the cancer dynamics. Parameters in the energy function refer the biochemical elements that induce cancer birth, growth, and metastasis; as well as the biochemical immune system process of defense.
Proper citation: Didier Barradas-Bautista, Matias Alvarado, Mark Agostino, Germinal Cocho 2018. Go game as metaphor of cancer metastasis. protocols.io dx.doi.org/10.17504/protocols.io.nqddds6 Copy
Authors: Emily E. Hare, Brant K. Peterson, Venky N. Iyer, Rudolph Meier, Michael B. Eisen
Group: VERVE Net
Summary: This protocol is from:Hare EE, Peterson BK, Iyer VN, Meier R, Eisen MB (2008) Sepsid even-skipped Enhancers Are Functionally Conserved in Drosophila Despite Lack of Sequence Conservation. PLoS Genet 4(6): e1000106. doi:10.1371/journal.pgen.1000106Please see the full manuscript for additional details.(Image: calf, Jim Champion, CC BY-SA)
Proper citation: Emily E. Hare, Brant K. Peterson, Venky N. Iyer, Rudolph Meier, Michael B. Eisen 2015. Eisen Lab, Fresh Cow Dung. protocols.io dx.doi.org/10.17504/protocols.io.ebebaje Copy
Authors: Tinka Beemsterboer, Lukas Snoek
Group: Spinoza Centre, REC-L
Summary: Dear researcher,This protocol gives you the steps to start an MRI project at the Spinoza Centre Roeterseiland. If you haven't had contact with Steven Scholte or Tinka Beemsterboer, make sure to contact them before completing this protocol. You can reach us by email:Steven Scholte: [email protected] Beemsterboer: [email protected] you have any suggestions for improving the workflow, don't hesitate to add comments to the steps in the protocol.Hope to see you soon!Spinoza REC Team!Steven Scholte, Tinka Beemsterboer en Lukas Snoek
Proper citation: Tinka Beemsterboer, Lukas Snoek 2018. Before Data Acquisition. protocols.io dx.doi.org/10.17504/protocols.io.jk5cky6 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the protocol for the Q5® Site-Directed Mutagenesis Kit (E0554)
Proper citation: New England Biolabs 2014. Q5® Site-Directed Mutagenesis (E0554). protocols.io dx.doi.org/10.17504/protocols.io.cjpumm Copy
Authors: David Dunigan and Irina Agarkova
Group: VERVE Net
Proper citation: David Dunigan and Irina Agarkova 2016. Chlorovirus Purification. protocols.io dx.doi.org/10.17504/protocols.io.er2bd8e Copy
Authors: Christelle Bouchard
Proper citation: Christelle Bouchard 2019. in situ hybridization performed on the peri-rhopalial tissue of a scyphozoan jellyfish. protocols.io dx.doi.org/10.17504/protocols.io.3qngmve Copy
Authors: Peter Kuhnert, Tobias Hidber
Summary: Dichelobacter nodosus is a gram-negative fastidious anaerobic bacterium and the causative agent of ovine footrot. The disease has a global presence and is endemic in many countries. Clinical symptoms range from mild interdigital dermatitis in benign footrot to severe underrunning and separation of the hoof horn from the underlying tissue in virulent footrot. Clinical symptoms start as early as 2 weeks after first contact and the disease leads to pain, lameness, decreased meat and wool production as well as animal welfare issues. Diagnosis has improved significantly by the development of a real-time PCR to detect and discriminate virulent (aprV2-positive) and benign (aprB2-positive) D. nodosus strains. However, the real-time PCR also detects dead cells making its use for testing e.g. disinfecting agents limited. A PMA (propidium monoazid) real-time PCR using the improved dye PMAxx™ was therefore developed for virulent D. nodosus that allows discrimination of viable and dead bacteria. The distinction between viable and non-viable cells is possible, based on membrane integrity. For that purpose, the samples containing D. nodosus are treated with the improved nucleic acid intercalating PMA dye PMAxx™ that selectively enters cells with compromised cell membranes, whereas the intact cell membrane presents a natural barrier for this molecule. After exposure to strong light, it covalently binds to the DNA, preventing DNA from being amplified by PCR, thereby enabling differentiation of viable from non-viable cells. The PMA-qPCR proved to be a valid method for comparison of antimicrobial efficiency in ex vivo experiments.
Proper citation: Peter Kuhnert, Tobias Hidber 2020. Detection of viable Dichelobacter nodosus by real-time PCR using PMAxx™. protocols.io dx.doi.org/10.17504/protocols.io.bbh9ij96 Copy
Authors: Richard Tennant, Paul Rutten
Group: iGEM Measurement
Summary: This is a protocol from the 2018 iGEM InterLab study which tests various 'standard' parts from the Registry.
Proper citation: Richard Tennant, Paul Rutten 2019. Standard iGEM Cell Measurement Protocol. protocols.io dx.doi.org/10.17504/protocols.io.zgmf3u6 Copy
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