Protocols   Select Another Resource Report Type

Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371)

Proper citation: New England Biolabs 2015. Dephosphorylation of 5´-ends of DNA using rSAP (M0371). protocols.io dx.doi.org/10.17504/protocols.io.crkv4v Copy   

•   Source: Protocols.io

Authors: Lumei Liu, Youngmi Koo, Boyce Collins, Zhigang Xu, Jagannathan Sankar, Yeoheung Yun
Summary: Melting and alloying operations were performed in a low carbon steel crucible in an inert ultra-high purity argon environment, preventing the oxidation reactions of molten Mg. High purity Mg (99.97%) was heated to 710°C for 10 minutes until the slug was completely melted. Each alloying element was added separately and kept for 10 minutes before additional alloy was added to ensure complete dissolution of all alloying elements. Automated stirring was applied for 15 minutes to ensure a homogeneous distribution of elements throughout the melt. The melt was then raised to 730 °C, held for 15 minutes and then poured into a steel tube mold. A solution heat treat was carried out on the as-cast alloy at 510 °C for 10 h before it was extruded. The extrusion was conducted with an extrusion ratio 10, extrusion temperature 400 ºC and ramp speed 1 mm/s.

Proper citation: Lumei Liu, Youngmi Koo, Boyce Collins, Zhigang Xu, Jagannathan Sankar, Yeoheung Yun 2017. ZWEK alloy fabrication procedure. protocols.io dx.doi.org/10.17504/protocols.io.iepcbdn Copy   

•   Source: Protocols.io

Authors: Sean Seaver
Summary: The working concentration of blasticidin S HCl for use in mammalian cell gene selection can range anywhere from 2-100μg/mL depending on the cell lines used.Optimal doses typically range from 2-20μg/mLHowever, we recommend performing a kill curve to determine the optimal dose for your cell lines.

Proper citation: Sean Seaver 2014. Blasticidin S HCl in Mammalian Culture Determine Working Concentration. protocols.io dx.doi.org/10.17504/protocols.io.cikucv Copy   

•   Source: Protocols.io

Authors: Josef Hoff
Summary: Preparation of electrocompetent cells Weinstock paper:Matthew T Weinstock, Eric D Hesek,Christopher M Wilson, Daniel G GibsonVibrio natriegens as a fast-growing host for molecular biology Nature Methods volume 13, pages 849–851 (2016)To prepare the day before: -LB I media +V2 salts (204 mM NaCl, 4.2 mM KCl, 23.14mM MgCl2 )-electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH 7) ( sterile filtrated)-over night culture of V.n from the cryo-stock in LBI + v2 salts (37 °C ; at 200 r.p.m)Preparing culture :Depending on how many aliquds you want to have, incubate media with your overnight culture for a starting OD of 0.05.The culture is grown at 37 °C in a baffled flask, shaking at 200 r.p.m. until an OD600 between 0.5 to 0.8 is reached. *be careful when they reach an OD near 0.1, V. natriegensis very fast growing, so start measuring in shorter time periods.Prechill the electroporation bufferWashing:From here on try always to keep your culture on ice.-The culture is then put on ice for 15 min. ( the original Protocol sais to directly fill them into your prechilled centrifugatin containments )-The cells are pelleted at 3000x g. for 20 min at 4 °C.-The supernatant is carefully decanted and the cell pellets are gently suspended in 10mL of chilled electroporation buffer.-The suspensions are transferred to a chilled50mL falcon tube and the tube is filled top with additional chilled electroporation buffer (50mL) and inverted several times. -The cells are centrifuged down at 3000x g for 15 min at 4 °C. -The wash is repeated two times for a total of three washes.Aliquotation:After the final wash, the supernant is carefully decanted the cells are gently resuspended in residual electroporation buffer. Measure the OD in a 1/20 dilution against electroporation buffer.The volume is adjusted with additional electroporation buffer to bring the final OD600 to 16.The Cells are aliquoted ( 80µL ) into chilled 1.5µL centrifugation tubes, directly frozen in liquid nitrogen and stored at −80 °C until use.

Proper citation: Josef Hoff 2018. Preparation of electrocompetent cells. protocols.io dx.doi.org/10.17504/protocols.io.u3heyj6 Copy   

•   Source: Protocols.io

Authors: Steven Burgess
Group: Ag SynBio Lab UIUC
Summary: This protein extraction protocol was developed for analysis of protein abundance in leaf tissue by immunoblot. It was optimized for dicot species including Glycine max.Quantities - 450 µL protein extraction buffer (PEB) per three 13.4 mm diameter leaf disks (size #7 Humboldt Cork Borer)Note from Agrisera: Keeping sample volumes in a range of 0.2-0.5 mL has been found to contribute to better extraction results, an upscale in volume is not recommended, if no cork borer available the suggestion is 500 µL PEB per 100 mg of plant tissue. - Final volume ~400 µL - Expected yield: 1.5 - 6 µg / µL - Total yield: 6 - 24 mg protein - Amount of total soluble protein to load per lane 30 µg Note: when using the TissueLyser II it is recommended to use 2 mL centrifuge tubes in conjunction with 4mm beads. 1.5mL tubes are narrow at the end and samples will not grind properly if you use the 4mm beads.

Proper citation: Steven Burgess 2020. Leaf Protein Extraction for Immunoblot (Soybean, Cowpea, Tobacco). protocols.io dx.doi.org/10.17504/protocols.io.bqnmmvc6 Copy   

•   Source: Protocols.io

Authors: Ruben M Costa, Anny Cárdenas, Christian Voolstra
Group: reefgenomics, Aiptasia-Symbiodiniaceae Model System
Summary: This protocol aims to obtain bacteria-depleted Aiptasia polyps. It is divided into 2 sections: (1) a rearing protocol composed of sterile food preparation and anemone priming and (2) a bacteria-depletion protocol for Aiptasia.

Proper citation: Ruben M Costa, Anny Cárdenas, Christian Voolstra 2019. Protocol for bacterial depletion of Aiptasia anemones - Towards the generation of gnotobiotic/germ-free cnidarian host animals. protocols.io dx.doi.org/10.17504/protocols.io.7fthjnn Copy   

•   Source: Protocols.io

Authors: Isabel Gautreau
Group: New England Biolabs (NEB)
Summary: Protocol for digestion with NEBNext dsDNA Fragmentase (M0348)

Proper citation: Isabel Gautreau 2015. Digestion with NEBNext dsDNA Fragmentase (M0348). protocols.io dx.doi.org/10.17504/protocols.io.cr4v8v Copy   

•   Source: Protocols.io

Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Appendix C of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of Vaccine Candidate against COVID-19 in adults > 18 years of age"This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.Please note that this is an evolving document, to be versioned and updated, based on community feedback and new data.

Proper citation: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores 2020. Sample Informed Consent Form Template (Appendix C of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io dx.doi.org/10.17504/protocols.io.bj6dkra6 Copy   

•   Source: Protocols.io

Authors: Elen Mello de Souza, Audrien Alves Andrade, Fernanda Cunha Jácome, Marcos Alexandre Nunes da Silva, Gabriela Cardoso Caldas, Ortrud Monika Barth, Debora Ferreira Barreto-Vieira
Summary: Zika Virus propagation

Proper citation: Elen Mello de Souza, Audrien Alves Andrade, Fernanda Cunha Jácome, Marcos Alexandre Nunes da Silva, Gabriela Cardoso Caldas, Ortrud Monika Barth, Debora Ferreira Barreto-Vieira 2017. Zika Virus propagation. protocols.io dx.doi.org/10.17504/protocols.io.jkackse Copy   

•   Source: Protocols.io

Authors: Bonnie Poulos
Group: VERVE Net, Sullivan Lab
Summary: Purpose: To test axenic cultures for purity.  Protocol described in S. Bertillson, O. Berglund, D.M. Karl, S.W. Chisholm (2003).  Elemental composition of marine Prochlorococcus and Synechococcus: Implications for the ecological stoichiometry of the sea.  Limnol Oceanogr 48(5):1721-1731.

Proper citation: Bonnie Poulos 2016. Marine Purity Broth. protocols.io dx.doi.org/10.17504/protocols.io.ekrbcv6 Copy   

•   Source: Protocols.io

Authors: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria Axmann
Group: Axmann Lab, CyanoWorld
Summary: This protocol describes how to detect synthesis of [α32P]ATP from [α32P]ATP by recombinant KaiC proteins. Radioactive nucleotides are separated via thin layer chromatography using TLC PEI Cellulose F plates as stationary phase and LiCl as soluble phase. The principle of this method is based on Egli et al. (Egli M, Mori T, Pattanayek R, Xu Y, Qin X, Johnson CH.2012. Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism. Biochemistry 51:1547-58.)

Proper citation: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria Axmann 2019. ATP synthase activity assay (radioactive). protocols.io dx.doi.org/10.17504/protocols.io.48qgzvw Copy   

•   Source: Protocols.io

Authors: Corentin Cras-Méneur
Summary: General fluorescent immunostaining protocol for paraffin sections

Proper citation: Corentin Cras-Méneur 2021. Fluorescent immunochemistry. protocols.io dx.doi.org/10.17504/protocols.io.brkvm4w6 Copy   

•   Source: Protocols.io

Authors: Thomas Mudersbach, Daniel Siuda, Karin Kohlstedt, Ingrid Fleming

Proper citation: Thomas Mudersbach, Daniel Siuda, Karin Kohlstedt, Ingrid Fleming 2019. Isolation of endothelial cells from umbillical vessels. protocols.io dx.doi.org/10.17504/protocols.io.ybdfsi6 Copy   

•   Source: Protocols.io

Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Summary: From Sullivan M., Lindell D., Lee J., Thompson L., Bielawski J., Chisholm S. Prevalence and Evolution of Core Photosystem II Genes in Marine Cyanobacterial Viruses and Their Hosts.PLOS Biology, 2006 4(8):e234. Please see the published manuscript for additional information.

Proper citation: Matthew Sullivan 2016. Amplification of phage genes from lysates and environmental samples. protocols.io dx.doi.org/10.17504/protocols.io.djn4md Copy   

•   Source: Protocols.io

Authors: Darach Miller
Summary: The aim of this protocol is to describe how to do a very reproducible upshift of a nitrogen-limited yeast culture to nitrogen non-limited conditions. In order to do this with enough cells to make inefficient analysis (ie RATEseq) feasible, we start in the chemostat for high-cell counts.

Proper citation: Darach Miller 2016. scratch, don't use, delete ( the biology is unexpected and something we don't want to invest time in figuring down right now ). protocols.io dx.doi.org/10.17504/protocols.io.fnnbmde Copy   

•   Source: Protocols.io

Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino
Summary: Published work using this protocol:- A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions.Alex Mabou Tagne, Franca Marino, Massimiliano Legnaro, Alessandra Luini, Barbara Pacchetti, Marco Cosentino. Int J Mol Sci. 2019 Apr 13;20(8):1833. doi: 10.3390/ijms20081833.- β 2 -Adrenoceptors Inhibit Neutrophil Extracellular Traps in Human Polymorphonuclear Leukocytes.Franca Marino, Angela Scanzano, Laura Pulze, Monica Pinoli, Emanuela Rasini, Alessandra Luini, Raffaella Bombelli, Massimiliano Legnaro, Magda de Eguileor, Marco Cosentino . J Leukoc Biol. 2018 Sep;104(3):603-614. doi: 10.1002/JLB.3A1017-398RR. Epub 2018 Apr 18.- Adrenergic Modulation of Migration, CD11b and CD18 Expression, ROS and interleukin-8 Production by Human Polymorphonuclear Leukocytes.Angela Scanzano, Laura Schembri, Emanuela Rasini, Alessandra Luini, Jessica Dallatorre, Massimiliano Legnaro, Raffaella Bombelli, Terenzio Congiu, Marco Cosentino, Franca Marino. Inflamm Res. 2015 Feb;64(2):127-35. doi: 10.1007/s00011-014-0791-8. Epub 2015 Jan 6.

Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PMN- 03 Culture of Human PMN - Migration. protocols.io dx.doi.org/10.17504/protocols.io.bhrmj546 Copy   

•   Source: Protocols.io

Authors: Li Kang
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Mice with catheters implanted in the jugular vein (infusions) and carotid artery (sampling) are used for this procedure. The hyperglycemic clamp is used to assess insulin secretory capacity in conscious mice in response to hyperglycemia. Plasma insulin and C-Pedtide concentrations are measured at various times during the 2-hr clamp period.

Proper citation: Li Kang 2019. Vandy - Hyperglycemic clamp. protocols.io dx.doi.org/10.17504/protocols.io.yxefxje Copy   

•   Source: Protocols.io

Authors: Julian J. Freen-van Heeren, Benot P. Nicolet, Monika C. Wolkers
Summary: A key feature of immune cells, such as T cells, is their rapid responsiveness to activation. The response rate of T cells depends on the type of signals they receive, and the signal strength. Studying the underlying mechanisms that define responsiveness, however, is confounded by the fact that immune cells do not uniformly respond to activation. Tools that measure gene products on a single cell level therefore provide additional insights in T cell biology. Here we describe Flow-FISH, a high-throughput assay that allows for the simultaneous measurement of cytokine mRNA and protein levels of the gene(s) of interest by flow cytometry. We present several possible applications of Flow-FISH in human and murine T cells that - with minor adjustments - should also be applicable for other gene products and cell types. This is a post-peer-review, pre-copyedit version of an article published in Methods in Molecular Biology, and is currently in press.

Proper citation: Julian J. Freen-van Heeren, Benot P. Nicolet, Monika C. Wolkers 2019. Combined Single-Cell Measurement of Cytokine mRNA and Protein in Immune Cells. protocols.io dx.doi.org/10.17504/protocols.io.3t9gnr6 Copy   

•   Source: Protocols.io

Authors: Caio Maximino, Denis Broock Rosembeg
Group: Fish behavior and physiology, Medicinal Plants Southeastern Pará Research Group
Summary: The protocol describes a spectrofluorophotometric method for rapid determination of monoamine oxidase (MAO) activity in zebrafish brains. The protocol is based on the transformation of kynuramine hydrobromide into 4-hydroxyquinoline. Since zebrafish possess only one MAO isoform, inhibitors of other isoforms are not necessary.

Proper citation: Caio Maximino, Denis Broock Rosembeg 2020. Monoamine oxidase activity in fish brain tissue . protocols.io dx.doi.org/10.17504/protocols.io.bgu9jwz6 Copy   

•   Source: Protocols.io

Authors: Elena I. Zuykova, Evgeniy P. Simonov, Nickolai A. Bochkarev, Sergey A. Abramov, Natalia G. Sheveleva, Alexey A. Kotov
Summary: Here we studied the genetic polymorphism of the ribosomal 12S and protein-coding ND2 mitochondrial genes in three closely related taxa of the Daphnia (Daphnia) longispina complex, namely D. galeata Sars, D. longispina O.F. Müller and D. dentifera Forbes, and performed an analysis of phylogenetic relationships between these taxa based on concatenated sequences of two genes.

Proper citation: Elena I. Zuykova, Evgeniy P. Simonov, Nickolai A. Bochkarev, Sergey A. Abramov, Natalia G. Sheveleva, Alexey A. Kotov 2018. untitled protocol. protocols.io dx.doi.org/10.17504/protocols.io.s7pehmn Copy   

•   Source: Protocols.io