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Authors: Britt Gratreak
Group: Ellison Lab
Summary: Basic protocol to achieve lipofectamine transfection of Human Embryonic Kidney 293 (HEK293) cells with cDNA and/or siRNA.
Proper citation: Britt Gratreak 2018. Cell Culture Transfection of HEK293 with cDNA and/or siRNA. protocols.io dx.doi.org/10.17504/protocols.io.nppddmn Copy
Authors: Zhenbin Hu, Geoff Morris
Group: TERRA Reference Phenotyping Platform
Summary: Select five plots as standard: 1 (weakest) - 5 (strongest), then other plots will be scored based on the similar with which standard plot.Seedling vigor is based on the size of seedling, greenness, uniformityScore each plot for seeding vigor was phenotyped by plotSeedling vigor was phenotyped every other day for six timesSeedling vigor phenotyping was started after 15 days of planting or 10 days after emergence
Proper citation: Zhenbin Hu, Geoff Morris 2019. Seedling vigor. protocols.io dx.doi.org/10.17504/protocols.io.jc2ciye Copy
Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Keyhole limpet hemocyanin (KLH) is a cooper-containing protein comprising of subunits with MW of 400 kDa. This protein is found in the hemolymph of the sea mollusk Megathura crenulata. It has the ability to enhance the host’s immune response by interacting with monocytes, T cells and macrophages. KLH has been used primarily as a carrier for vaccines and antigens [1]. It was found that chicken immunized with KLH bound peptide raised an anti-KLH immunoresponse [2]. This can be tested by a single method such as the Ouchterlony technique.Reference1. Aarntzen EH, de Vries IJ, Göertz JH, et al. Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters.Cancer Immunol Immunother. 2012;61(11):2003-2011. doi:10.1007/s00262-012-1263-z2. Justiz Vaillant AA, Anderson MF, Smikle M, Wisdom B, Mohammed W, et al. (2013) Development of Anti HIV Gp120 and HIV Gp41 Peptide Vaccines. J Vaccines Vaccin 4: 206. doi: 10.4172/2157-7560.1000206
Proper citation: Angel Justiz-Vaillant 2020. Detection of anti- keyhole limpet hemocynin (anti-KLH) antibodies by double immunodiffusion (Ouchterlony) technique.. protocols.io dx.doi.org/10.17504/protocols.io.bjspkndn Copy
Authors: Rob Lampe, Andrew Allen
Group: A.E. Allen Lab
Summary: A mostly automated protocol for extraction of genomic DNA from seawater filtered onto a Sterivex filter (Cat. No. SVGP0150). Reagents come from the Macherey-Nagel NucleoMag Plant Kit for DNA purfication (Cat. No. 744400). Automated liquid handling is performed on an eppendorf EpMotion 5075t with multi-channel pipettes. This protocol assumes that the Sterivex is sealed with tube sealant on the male end and a male-luer lock plug on the female end. Using this protocol, we routinely extract ~80 samples at a time.The protocol has been modified to increase to an 800 µL starting volume. This normally results in less Binding Buffer MC2 than is needed. Extra binding buffer should be purchased separately. The kit manual can be found here: https://www.mn-net.com/media/pdf/09/46/a0/Instruction-NucleoMag-Plant.pdfThe epMotion program is attached here.
Proper citation: Rob Lampe, Andrew Allen 2020. Sterivex DNA extraction. protocols.io dx.doi.org/10.17504/protocols.io.bcysixwe Copy
Authors: Fang He, Aiting Zhou, Shuo Feng
Proper citation: Fang He, Aiting Zhou, Shuo Feng 2018. Human amniotic epithelial cells in mouse bleomycin lung fibrosis models: a systematic review and meta-analysis. protocols.io dx.doi.org/10.17504/protocols.io.pjqdkmw Copy
Authors: Andrea Málková, Ales Tichy
Summary: Dicentric Chromosomes Assay
Proper citation: Andrea Málková, Ales Tichy 2018. Dicentric Chromosome Assay. protocols.io dx.doi.org/10.17504/protocols.io.mjgc4jw Copy
Authors: ZengU19 BRAIN grant
Group: BICCN, Allen Institute for Brain Science
Summary: Protocol to generate full-length cDNA from single cells, or nuclei, using Takara SMARTer V4.
Proper citation: ZengU19 BRAIN grant 2019. SMARTerV4 (1x) Amplification for single-cell or single-nuclei RNASeq Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7d6hi9e Copy
Authors: Pierre Teodosio
Proper citation: Pierre Teodosio 2020. AMOVA AND PHILOGENY IN SARS-COV- 2 PROTOCOLS. protocols.io dx.doi.org/10.17504/protocols.io.bixjkfkn Copy
Authors: Girija Goyal
Summary: Commonly used protocol to isolate peripheral blood mononuclear cells from whole human blood or apheresis packs
Proper citation: Girija Goyal 2018. PBMC Isolation from apheresis collars with SepMate tubes. protocols.io dx.doi.org/10.17504/protocols.io.qpcdviw Copy
Authors: Jie Deng, Hua Gao, Zhen Gao, Huaxian Zhao, Ying Yang, Qiaofen Wu, Bo Wu, Chengjian Jiang *
Summary: Methods and procedures appeared in this study.
Proper citation: Jie Deng, Hua Gao, Zhen Gao, Huaxian Zhao, Ying Yang, Qiaofen Wu, Bo Wu, Chengjian Jiang * 2017. Identification and molecular characterization of a metagenome-derived L-lysine decarboxylase gene from subtropical soil microorganisms. protocols.io dx.doi.org/10.17504/protocols.io.jdaci2e Copy
Authors: Ben Tully
Group: ECOGEO
Summary: Quality impact results is the universal first step for all sequencing methods. Sequencing results distrputed in FASTQ format.
Proper citation: Ben Tully 2016. Quality Assessment: FastQC. protocols.io dx.doi.org/10.17504/protocols.io.fa3bign Copy
Authors: Openwetware
Summary: Nucleic acid precipitation is used to concentrate and/or purify nucleic acids. The below protocol is based on the fact that nucleic acids are less soluble in alcohol than in more polar water. Addition of salt further decreases solubility by competing for water dipoles; as does low temperature. Please see the OpenWetWare website for more details.Citation: OpenWetWare contributors, 'Ethanol precipitation of nucleic acids', OpenWetWare, , 1 July 2012, 10:48 UTC, http://openwetware.org/index.php?title=Ethanol_precipitation_of_nucleic_acids&oldid=611420> [accessed 21 January 2015]
Proper citation: Openwetware 2015. Ethanol precipitation of nucleic acids (Eppendorf tubes). protocols.io dx.doi.org/10.17504/protocols.io.cdrs55 Copy
Authors: Michael Cianfrocco
Group: Cianfrocco Lab @ University of Michigan
Summary: Below is a protocol for logging in and submitting a RELION GPU job to Comet GPU nodes at the San Diego Supercomputer Center.
Proper citation: Michael Cianfrocco 2017. Submitting RELION GPU jobs to Comet at SDSC. protocols.io dx.doi.org/10.17504/protocols.io.i3ecgje Copy
Authors: Ben Kuipers
Group: iGEM Wageningen 2019
Summary: Mix and Go protocol for the preparation of competent E. coli cells. (This protocol is according to Zymo Research mix and Go protocol).
Proper citation: Ben Kuipers 2019. Mix and go competent cells. protocols.io dx.doi.org/10.17504/protocols.io.7jnhkme Copy
Authors: Eugene Drokhlyansky, Nicholas Van Wittenberghe, Michal Slyper, Julia Waldman, Asa Segerstolpe, Orit Rozenblatt-Rosen, Aviv Regev
Group: NCIHTAN
Summary: This protocol describes a method based on work by Drokhlyansky et al. for nuclei isolation from frozen tissue. It can be used on both healthy and disease tissues and is compatible with droplet-based single-nucleus RNA-Seq technology Slyper et al.This method is part of a toolbox for processing frozen tissue samples for single-nucleus RNA-Seq, including the CST (this protocol), TST and NST protocols (all available in protocols.io). We recommend users to test all three protocols on their tissue type of interest and perform side-by-side comparison of the data generated. A description of the complete toolbox and guidance for testing and selecting methods from the toolbox for processing other tumors can be found in Slyper et al.For the Human Tumor Atlas Pilot Project (HTAPP), the CST-nuclei isolation method was used to profile pediatric glioma frozen samples. It was also tested on metastatic breast cancer, ovarian cancer, pediatric sarcoma, and pediatric neuroblastoma frozen samples. For these cancer types, however, TST was the protocol of choice.
Proper citation: Eugene Drokhlyansky, Nicholas Van Wittenberghe, Michal Slyper, Julia Waldman, Asa Segerstolpe, Orit Rozenblatt-Rosen, Aviv Regev 2020. HTAPP_CST- Nuclei isolation from frozen tissue. protocols.io dx.doi.org/10.17504/protocols.io.bhbej2je Copy
Authors: Elizabeth Neumann, Jamie Allen, Jeff Spraggins, Danielle Gutierrez
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope: Annotate lipid species detected by MALDI IMS analysis.Expected Outcome:A list of lipid identifications with low mass errors (
Proper citation: Elizabeth Neumann, Jamie Allen, Jeff Spraggins, Danielle Gutierrez 2020. Lipid Annotation of MALDI IMS Datasets. protocols.io dx.doi.org/10.17504/protocols.io.864hzgw Copy
Authors: Bonnie Hurwitz, Ken Youens-Clark
Group: VERVE Net, Hurwitz Lab
Summary: PCPipe is a protein-clustering tool. The input is a set of ORFs and a FASTA file with already clustered ORFs. The process entails:Use cd-hit-2d to compare the input peptides to previously clustered proteinsThe result is a file with input proteins that clustered to existing clusters and those that did notUse the unclustered peptides and self-cluster them via cd-hitTake a representative sequence from each novel cluster, and use "blastp" to compare to SIMAP.Use the resulting SIMAP "feature_id" to look up the SIMAP features, merging the query results with the protein ID into a tab-delimited annotations fileProvide the user with two cluster files and the annotations for the new clusters based on the representative sequenceCode is freely available at Github.
Proper citation: Bonnie Hurwitz, Ken Youens-Clark 2016. PCPipe: Protein clustering with SIMAP annotations. protocols.io dx.doi.org/10.17504/protocols.io.ehfbb3n Copy
Authors: Omar Valencia
Summary: Evaluation of β-diversity partitioning in its spatial turnover (BJTU) and nestedness (BJNE) components along a latitudinal gradient across biogeographic provinces.
Proper citation: Omar Valencia 2018. Gamma-diversity partitioning of gobiid fishes (Teleostei: Gobiidae) ensemble along of Eastern Tropical Pacific: biological inventory, latitudinal variation and species turnover. protocols.io dx.doi.org/10.17504/protocols.io.sbbeain Copy
Authors: Bioline
Summary: The ISOLATE II Biofluids RNA Kit is specially developed for the rapid phenol-free isolation of high quality total RNA from biofluids and viruses. Total RNA can be purified from blood, plasma, serum and other types of biofluids such as saliva, urine, semen and cerebrospinal fluid (CSF). Viral RNA can be isolated from the same sample sources, as well as cultured cells and tissue. The kit isolates all sizes of RNA from large mRNA, viral RNA and ribosomal RNA (rRNA) down to small RNAs such as microRNA (miRNA) and short interfering RNA (siRNA).
Proper citation: Bioline 2016. ISOLATE II Biofluids RNA Kit. protocols.io dx.doi.org/10.17504/protocols.io.f5kbq4w Copy
Authors: Steven Henikoff, Jorja Henikoff, Hatice Kaya-Okur, Kami Ahmad
Group: Human Cell Atlas Method Development Community
Summary: CUT&Tag@direct uses a modification of Bench-top CUT&Tag which includes incubation in 0.1% SDS post-tagmentation for quantitative release of targeted fragments, followed directly by PCR with Triton-X100 to neutralize the SDS. This protocol is performed in single PCR tubes from nuclei to sequencing-ready libraries and should be suitable for high throughput. The protocol has been enhanced by the addition of hyperaccessibility mapping by Cleavage Under Targeted Accessible Chromatin (CUTAC), where H3K4me2 CUT&Tag samples are tagmented in low salt for mapping of the hyperaccessible site close to the H3K4me2-labeled nucleosomes.In situ tethering for CUT&Tag chromatin profiling.a) The steps in CUT&Tag. Added antibody (green) binds to the target chromatin protein (blue) between nucleosomes (gray ovals) in the genome, and the excess is washed away. A second antibody (orange) is added and enhances tethering of pA-Tn5 transposome (gray boxes) at antibody-bound sites. After washing away excess transposome, addition of Mg++ activates the transposome and integrates adapters (red) at chromatin protein binding sites. After DNA purification genomic fragments with adapters at both ends are enriched by PCR. b) CUT&Tag is performed on a solid support. Unfixed cells or nuclei (blue) are permeabilized and mixed with antibody to a target chromatin protein. After addition and binding of cells to Concanavalin A-coated magnetic beads (M), all further steps are performed in the same reaction tube with magnetic capture between washes and incubations, including pA-Tn5 tethering, integration, and DNA purification.
Proper citation: Steven Henikoff, Jorja Henikoff, Hatice Kaya-Okur, Kami Ahmad 2020. CUT&Tag-direct with CUTAC. protocols.io dx.doi.org/10.17504/protocols.io.bmbfk2jn Copy
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