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Authors: Wen Aw
Group: Cage Studies
Proper citation: Wen Aw 2018. Mitochondrial oxygen consumption. protocols.io dx.doi.org/10.17504/protocols.io.rrud56w Copy
Authors: Ramon Terrado
Group: Caron Lab - Protistan Ecology
Summary: This protocol describes how to isolate bacteria in order to label its biomass using 13C-glucose and 15N-ammonia. The first steps aim to isolate candidate bacteria that are able of growing on the chosen substrates for isotopic labeling.The protocol was applied to the isolation of bacetria from freshwater algal cultures, although it can be applied to natural samples.The M9 media used in the protocol is based on the media from Marley, Lu and Bracken (2001).References:Marley, J., Lu, M., & Bracken, C. (2001). A method for efficient isotopic labeling of recombinant proteins. Journal of Biomolecular Nmr, 20(1), 71–75.
Proper citation: Ramon Terrado 2016. Isolation and isotopic labeling of bacteria. protocols.io dx.doi.org/10.17504/protocols.io.eyzbfx6 Copy
Authors: Sebastian Triesch
Group: M4455 - Synthetische Biologie und Biotechnologie
Summary: This protocol is under development and for teaching purposes only!Nile Red is a fluorescent dye that stains selectively hydrophobic substances. We assume that squalene accumulates in the cell membrane or in lipid vesicles where it can be stained by the dye.
Proper citation: Sebastian Triesch 2019. Squalene Quantification using Nile Red Staining (M4455 Version). protocols.io dx.doi.org/10.17504/protocols.io.y6vfze6 Copy
Authors: Anna Behle
Group: Axmann Lab
Summary: This protocol covers the inoculation, cultivation, IPTG-induction and plate reader measurement of E. coli BL21 strains carrying constructs with various toehold switch devices from Green et al., 2014.
Proper citation: Anna Behle 2017. Plate Reader experiment. protocols.io dx.doi.org/10.17504/protocols.io.kx5cxq6 Copy
Authors: Susanna C. Concilio
Proper citation: Susanna C. Concilio 2020. Determination of infectious lentiviral titers with GFP-encoding lentivirus. protocols.io dx.doi.org/10.17504/protocols.io.bcbsisne Copy
Authors: Grace Burgin
Summary: coating standard blank slides with poly-d lysine to increase adhesion of tissue
Proper citation: Grace Burgin 2019. Poly-d lysine coating slides. protocols.io dx.doi.org/10.17504/protocols.io.32cgqaw Copy
Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: The first step in any MERFISH experiment is the design of the oligonucleotide probes that will be used to label individual RNA species. In our current implementation of MERFISH, each oligonucleotide encoding probe consists of three basic components as illustrated in Figure 2. The first region is a 30-nt targeting region that is complementary to a portion of the sequence of the RNA to which it is designed to bind. The second region is a set of sequences that are called readout sequences, which were designed to be complementary and hence only bind to MERFISH readout probes and not other nucleic acid in the cell. Finally, the third region is a set of priming regions used in the construction of these probes, which will be discussed in detail in Probe Construction. In addition to the nucleotide sequences for each of these components, a codebook —the specific set of binary barcodes that will be used and their association with different RNA species of interest—must also be designed. In this section, we provide protocols to design these sequences and to build a codebook. Example code to perform these steps can be found at http://zhuang.harvard.edu/merfish/ .
Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Design of Oligonucleotide Probes. protocols.io dx.doi.org/10.17504/protocols.io.menc3de Copy
Authors: Kelsey Knight
Group: BioLegend
Summary: Reagents - Basic cell culture media: respective to the cell line used and experimental design Distilled water: Invitrogen® UltraPure™ Distilled Water (catalog number 19977015) or equivalent product from other vendor.Highthroughput (HT) Pro A or G 96 well plate or Spin Column (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)96-well collection plate: USA scientific® TempPlate non-skirted 96-well PCR plate natural (catalog number 1402-9596) or equivalent product from other vendor.QIAquick™ PCR purification kit (Qiagen®; catalog number 28104) or equivalent product from other vendor.Hypotonic Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Digestion Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Lysis Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Column Conditioning Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Wash Buffer 1 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Wash Buffer 2 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Wash Buffer 3 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Elution Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)5M NaCl: dissolve 292g of NaCl in 800mL of H2O. Adjust the volume to 1L with H2O.1M NaHCO3: dissolve 12.6g of NaHCO3 in 100 mL of H2O. Adjust the volume to 150 mL with H2O.Protease Inhibitor Cocktail (PIC): Cell signaling Technology® (catalog number 5871) or equivalent product from other vendor.Proteinase K: Qiagen® (catalog number 19133) or equivalent product from other vendor.Proteinase K stop solution: 100mM Phenylmethylsulfonyl fluoride (PMSF). Weigh 1.742g PMSF (Sigma-Aldrich®, catalog number 10837091001) and add DMSO (Sigma-Aldrich®, catalog number D2650) to a final volume of 100mL. Dissolve completely.PBS: HyClone™ Phosphate Buffered Saline (1X) (catalog number SH30256.01) or equivalent product from other vendor.Formaldehyde: VWR® 37% w/w aqueous solution (catalog number 97064-604) or equivalent product from other vendor.0.65M Glycine: Dissolve 488g of glycine into 1L of H2O to make 6.5M glycine (10X). Dilute with PBS to make 1X before each experiment. Micrococcal Nuclease: Cell Signaling Technologies® (catalog number 10011S) or equivalent product from other vendor.1.3-1.5% Agarose gel: dissolve 1.3-1.5g of agarose powder into 100mL of TAE buffer by microwave heating. Visualize DNA under UV light by adding appropriate amount of Ethidium Bromide, or use an equivalent method.Add appropriate amount of Ethidium Bromide to cooled down gel solution to visualize DNA under UV light.0.5M EDTA: Dissolve 186.1g EDTA into 700mL of H2O. Adjust the volume to 1L with H2O.100bp ladder: NEB® (catalog number N0467S) or equivalent product from other vendor.Materials -Heat blockRotatorCentrifugeMicrocentrifuge37ºC incubatorNanodropSonicatorUV light imagerRocking platform
Proper citation: Kelsey Knight 2018. Chromatin Immunoprecipitation (ChIP) Assay Protocol. protocols.io dx.doi.org/10.17504/protocols.io.tj7ekrn Copy
Authors: Andrew Crowley
Group: Ackerman Lab
Summary: Protocols for the amine coupling of fluorescent microspheres with antigen or capture reagent for opsonization and phagocytosisQuantity as written: approx. 1 x 108 under typical yields (enough for 400 phagocytosis wells under standard protocol)
Proper citation: Andrew Crowley 2019. Phagocytosis Bead Conjugation. protocols.io dx.doi.org/10.17504/protocols.io.4cygsxw Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the preparation of a fixed mouse brain specimen bonded to an agarose block for blockface imaging and sectioning using the TissueCyte 1000 system.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. TissueCyte Specimen Embedding Acrylamide Coembedding. protocols.io dx.doi.org/10.17504/protocols.io.bf65jrg6 Copy
Authors: Marijn Ceelen
Group: iGEM Wageningen 2019
Summary: A protocol for phage lambda and phage T7 phage stock preparation
Proper citation: Marijn Ceelen 2019. Phage stock preparation. protocols.io dx.doi.org/10.17504/protocols.io.7kthkwn Copy
Authors: James M. Burnette III, Susan R. Wessler
Group: Genetics
Summary: This protocol is based on one described by Li et al. (2010) and has been modified to work in a college laboratory setting. The protocol is from:James M. Burnette III andSusan R. Wessler (2013) Transposing from the Laboratory to the Classroom to Generate Authentic Research Experiences for UndergraduatesGenetics193:367-375; doi:10.1534/genetics.112.147355Students need approximately 1.5 hours to extract DNA from up to five samples and the protocol can be carried out over several class periods by stopping at steps 6 and 9. Please see the full manuscript for additional details.
Proper citation: James M. Burnette III, Susan R. Wessler 2015. DNA Extraction for college laboratory setting. protocols.io dx.doi.org/10.17504/protocols.io.dwu7ev Copy
Authors: RACHELE CESARONI
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: RACHELE CESARONI 2017. Euplotes crassus transformation using Lipofectamine 2000 as vehicle. protocols.io dx.doi.org/10.17504/protocols.io.g5aby2e Copy
Authors: Robbie M. Martin, Steven W. Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: This protocol is an acid-phenol-based method for extracting RNA from samples collected onto polycarbonate filters. Bead-beating is included to increase yields from difficult to lyse cells, for example, in some species of cyanobacteria. We have used it successfully in RNA-sequencing projects involving lab cultures of cyanobacteria and from freshwater and marine environmental samples.
Proper citation: Robbie M. Martin, Steven W. Wilhelm 2020. Phenol-based RNA extraction from polycarbonate filters. protocols.io dx.doi.org/10.17504/protocols.io.bivuke6w Copy
Authors: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria Axmann
Group: CyanoWorld
Summary: This protocol can be used to investigate protein-protein interaction via yeast two-hybrid experiments. It describes the yeast-two hybrid method relying on the activity of the histidine-synthetase. If the proteins interact, the yeast cells are able to grow on selective medium without histidine.
Proper citation: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria Axmann 2019. Protein interaction analysis of KaiC3 with various Kai homologs via yeast two-hybrid experiments (Growth Assay). protocols.io dx.doi.org/10.17504/protocols.io.wcnfave Copy
Authors: Rini Varghese, Jasmine Ma, James Gordon, Robert L Sainburg, Carolee J Winstein
Summary: Successful bimanual coordination is accomplished by overcoming a variety of cognitive, perceptual and neuromotor constraints, e.g. higher-order representation of task goals, visual perceptual demands, attention demands, task demands (i.e., speed, accuracy) and sensorimotor integration. Sensorimotor interference in planning, executing and correcting bilateral movements especially arise when the hands are required to accomplish two independent or disparate goals. In this study, we describe the experimental paradigm to study sensorimotor interference during performance of a planar reaching task, in which the experimental condition consists of the two hands moving to two visually cued targets at separate distances (spatially asymmetric). Whereas the control conditions consisted of same-size movements performed unimanually with each limb and bimanually to symmetric targets. The paradigm is a modification of a previously conceptualized model task paradigm (e.g. 1-5) to study interference in discrete bilateral movements, wherein the two limbs are tasked with accomplishing asymmetric movement goals. However, our study paradigm involved important modifications to task design such as absence of online visual feedback (so as to test fairly rapid preprogrammed movements) and task instructions which emphasized speed and accuracy but not interlimb synchrony. The study aims: 1) to characterize sensorimotor interference in discrete bilateral reaching movements in young adults, and subsequently, 2) to determine the influence of the side of stroke on such sensorimotor interference. The experimental paradigm was conducted using the KineReach kinematic data collection system and accompanying software (Sainburg, Pennsylvania State University). MATLAB code to generate session and target files for this experiment are included in this protocol. Details of data processing and analysis will be published in the primary papers and code will be made available thereafter.References:1. Kelso (1979)2. Marteniuk (1984)3. Swinnen (1991)4. Diedrichsen (2006)5. Blich (2011)
Proper citation: Rini Varghese, Jasmine Ma, James Gordon, Robert L Sainburg, Carolee J Winstein 2020. Bimanual Interference Experimental Paradigm. protocols.io dx.doi.org/10.17504/protocols.io.7r3hm8n Copy
Authors: Adolfo Maria TAMBELLA, Evelina SERRI, Fulvio LAUS, Anna Rita ATTILI
Summary: A step-by-step procedure for autologous platelet-rich plasma production was developed for topical percutaneous injection in donkeys.This protocol was used in the following publication:Faillace V, Tambella AM, Fratini M, Paggi E, Dini F, Laus F. Use of autologous platelet-rich plasma for a delayed consolidation of a tibial fracture in a young donkey. The Journal of Veterinary Medical Science, 79(3), 2017: 618-622. (ISSN: 0916-7250) (DOI: 10.1292/jvms.16-0400)https://doi.org/10.1292/jvms.16-0400https://www.jstage.jst.go.jp/article/jvms/79/3/79_16-0400/_pdf/-char/en
Proper citation: Adolfo Maria TAMBELLA, Evelina SERRI, Fulvio LAUS, Anna Rita ATTILI 2018. Injectable autologous platelet-rich plasma for regenerative medicine in donkeys. protocols.io dx.doi.org/10.17504/protocols.io.ncbdasn Copy
Authors: Eva L. Feldman
Group: Mouse Metabolic Phenotyping Centers
Summary: Intra-epidermal nerve fiber density (IENFD) is used as a tool to assess small fiber neuropathy.
Proper citation: Eva L. Feldman 2019. U Michigan - Intra-Epidermal Fiber Density. protocols.io dx.doi.org/10.17504/protocols.io.563g9gn Copy
Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Proper citation: Angel Justiz-Vaillant 2020. Immunoblot analyses for investigating SpLA binding to purified mammalian and avian immunoglobulins.. protocols.io dx.doi.org/10.17504/protocols.io.bjq7kmzn Copy
Authors: Stephanie Loeb, Katy Graham, Marlene Wolfe, Krista Wigginton, Alexandria Boehm
Group: Coronavirus Method Development Community, Wastewater-based epidemiology working group
Summary: OverviewThis SOP describes pre-analytical procedures to be followed for the isolation and identification of SARS-CoV-2 RNA in primary settled solids samples from wastewater treatment plants. This protocal should be paired with our digital PCR protocal. The protocol follows the approximate workflow:Separate and aliquot sample (~4 hrs)Spike with BCoV (~1 hr)Extract RNA (~ 12 hours total, may be split into 2-3 steps/days)Inhibitor removal (~1 hr)Biosafety ConcernsConcentration and extraction procedures that utilize raw samples must adhere to strict Biosafety Level 2+ procedures. These procedures should be performed in a dedicated room. Downstream products may be handled using standard laboratory safety guidelines. This protocol was developed by Stephanie Loeb, Katy Graham, Marlene Wolfe, Krista Wigginton, and Alexandria Boehm at Stanford University and University of Michigan.
Proper citation: Stephanie Loeb, Katy Graham, Marlene Wolfe, Krista Wigginton, Alexandria Boehm 2020. Extraction of RNA from Wastewater Primary Solids Using a Direct Extraction Method for Downstream SARS-CoV-2 RNA Quantification. protocols.io dx.doi.org/10.17504/protocols.io.bi6skhee Copy
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