Searching the RRID Resource Information Network
Are you sure you want to leave this community? Leaving the community will revoke any permissions you have been granted in this community.
Authors: Jernej Turnsek, Pardis Gholami
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol presents a Gibson Assembly design for highly efficient construction of diatom episomes. We regularly observe >90% efficiency (efficiency = % of screened bacterial colonies containing the desired construct) following the steps presented here.
Proper citation: Jernej Turnsek, Pardis Gholami 2017. Guidelines for highly efficient construction of diatom episomes using Gibson Assembly . protocols.io dx.doi.org/10.17504/protocols.io.jy7cpzn Copy
Authors: Dan Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: February, 2012, based on RNAqueous May 29, 2008 protocol revision C, TURBO DNA-free June 9, 2009 protocol 1907M revision F, phenol/chloroform protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5438.full), ethanol precipitation protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5440.full)
Proper citation: Dan Richter 2017. RNAqueous with DNAse Clean-up by Phenol:Chloroform. protocols.io dx.doi.org/10.17504/protocols.io.iskcecw Copy
Authors: Yoshihisa Hirakawa
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Yoshihisa Hirakawa 2016. Transformtion of Perkinsus marinus by Amaxa and Bio-Rad. protocols.io dx.doi.org/10.17504/protocols.io.e5nbg5e Copy
Authors: Francois-Yves bouget
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes the selection and growth of stable transformants in semi solid agarose medium. Developped initially for Ostreococcus tauri, it also works for Bathycoccus.
Proper citation: Francois-Yves bouget 2017. Selection of stable transformants in Ostreococcus tauri and Bathycoccus prasinos. protocols.io dx.doi.org/10.17504/protocols.io.hc4b2yw Copy
Authors: Elena Casacuberta, Aleksandra Kozyczkowska, Sebastian Najle
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology
Summary: This is a protocol for transient transfection of the Icthyosporean Abeoforma whisleri. This protocol has an efficiency of 1-2% calculated from fluorescent positive cell from Flow Cytometry data, using the amount of cells and DNA described in the protocol. Increasing DNA from above 1 microgram/microliter does not increase transfection.
Proper citation: Elena Casacuberta, Aleksandra Kozyczkowska, Sebastian Najle 2019. Abeoforma whisleri transient transfection protocol. protocols.io dx.doi.org/10.17504/protocols.io.zexf3fn Copy
Authors: Fatma Gomaa, Zhou Zhuha, Roberto Docampo, Virginia Edgcomb, Peter Girguis
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Fatma Gomaa, Zhou Zhuha, Roberto Docampo, Virginia Edgcomb, Peter Girguis 2018. Bodo saltans Cassette for tagging EF1alpha gene__IG BsTub. protocols.io dx.doi.org/10.17504/protocols.io.s5jeg4n Copy
Authors: Daniel Richter, Parinaz Fozouni, Michael Eisen, Nicole King
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, King Lab, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: The origin of animals, which occurred over 600 million years ago, left no evidence in the fossil record. To trace the earliest events in animal prehistory, we compare extant animals to their closest living relatives, the choanoflagellates, in order to reconstruct the gene content of their last common ancestor and how it evolved on the stem lineage leading to animals. In this project, we increase the accuracy of ancestral animal and choanoflagellate gene content reconstructions by sequencing the transcriptomes of 19 species of choanoflagellates selected for their phylogenetic diversity.
Proper citation: Daniel Richter, Parinaz Fozouni, Michael Eisen, Nicole King 2017. Transcriptome sequencing of 19 diverse species of choanoflagellates. protocols.io dx.doi.org/10.17504/protocols.io.kwscxee Copy
Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol was used to express a nourseothricin (nou/nat) resistance gene and a gene encoding a silaffin precursor TpSil3p-APEX2 fusion protein in Thalassiosira pseudonana (Tp) strain CCMP1335 using conjugation by largely following Karas et al. (2015). In addition to the protocol, I am listing some of my observations working with this diatom species. Please check the 'Before start' and 'Guidelines' sections for more information including a list of Tp episomes I've constructed and which are available upon request. For additional background on my overacrching experimental aim, please refer to this document. 1. E. V. Armbrust, The Genome of the Diatom Thalassiosira Pseudonana: Ecology, Evolution, and Metabolism. Science. 306, 79–86 (2004).2. B. J. Karas et al., Designer diatom episomes delivered by bacterial conjugation. Nat. Commun. 6, 6925 (2015).Keeping in mind that diatom episomes contain a yeast centromere, it would be very interesting to see if conjugation could be adapted to organisms beyond diatoms.
Proper citation: Jernej Turnsek 2016. Conjugation of Thalassiosira pseudonana. protocols.io dx.doi.org/10.17504/protocols.io.f55bq86 Copy
Authors: Dan Needleman
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Dan Needleman 2017. Protocol for drug sensitivity assay with S. robusta. protocols.io dx.doi.org/10.17504/protocols.io.g9ebz3e Copy
Authors: Angela Piersanti, Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Euplotes crassus GFP-neo artificial nanochromosomes
Proper citation: Angela Piersanti, Rachele Cesaroni 2019. Euplotes crassus GFP-neo artificial nanochromosomes sequence. protocols.io dx.doi.org/10.17504/protocols.io.2atgaen Copy
Authors: G Jason Smith, April Woods
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Environmental Biotechnology Lab
Summary: This is a modified version of 'Conjugation of Thalassiosira pseudonana', publishedby J. Turnsek dx.doi.org/10.17504/protocols.io.f55bq86 .The protocol was modified to enhance viability in the diatom Pseudo-nitzschia multiseries. This version was used to transform Pseudo-nitzschia multiseries isolate 15091C3 with an episomal plasmid derived from pPtPUC3 to express a egfp gene under control of the P. multiseries actin promoter and termination domains. PLASMID INFOpPmAGFPC10 EPI : A P. multiseries derived expression cassette under control of P. multiseries ca 1000 bp of actin promoter and termination domains. Pm actPs::eGFP::actTs
Proper citation: G Jason Smith, April Woods 2019. Modified Bacterial Conjugation Protocol For Pseudo-nitzschia multiseries. protocols.io dx.doi.org/10.17504/protocols.io.7vhhn36 Copy
Authors: Binnypreet Kaur
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes
Summary: Cas9 Nuclease, S. pyogenes, (Cas9) is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA in vitro using Cas9 and a single guide RNA (sgRNA).
Proper citation: Binnypreet Kaur 2018. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386). protocols.io dx.doi.org/10.17504/protocols.io.rmud46w Copy
Authors: Lu Wang, Brittany Sprecher, Huan Zhang, Senjie Lin
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Protocol of dinoflagellate cell transformation
Proper citation: Lu Wang, Brittany Sprecher, Huan Zhang, Senjie Lin 2019. Dinoflagellate transformation. protocols.io dx.doi.org/10.17504/protocols.io.7prhmm6 Copy
Authors: Jackie L. Collier
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: Modified from Lippmeier et al. 2009
Proper citation: Jackie L. Collier 2018. Labyrinthulomycete DNA extraction protocol. protocols.io dx.doi.org/10.17504/protocols.io.n83dhyn Copy
Authors: Lev Tsypin, Aaron Turkewitz
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes how we grew S. robusta and stored cultures at 4 C. Under these condtiions, the cells grow to a maximum density of 2-2.5 x 106 cells/mL.
Proper citation: Lev Tsypin, Aaron Turkewitz 2019. Cultivation of Seminavis robusta. protocols.io dx.doi.org/10.17504/protocols.io.337gqrn Copy
Authors: David Booth
Group: King Lab, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol details the preparation and execution of CRISPR/Cas9 genome editing in S. rosetta. The protocol builds on a method to transfect macromolecules into S. rosetta for delivering a purified Cas9 ribonucleoprotein from Streptomyces pyogenes (SpCas9 RNP) into S. rosetta. Upon cleaving the S. rosetta genome at locations specified by the guide RNA (gRNA) of the SpCas9 RNP, S. rosetta can use DNA oligonucleotides as templates to repair the double-stranded break. Those repair templates can encode foreign sequences and mutations for editing the S. rosetta genome, so long as DNA oligonucleotides have >30 bases of sequence that is homologous to both sides of the Cas9 cleavage site.
Proper citation: David Booth 2020. Genome editing in the choanoflagellate Salpingoeca rosetta. protocols.io dx.doi.org/10.17504/protocols.io.89fhz3n Copy
Authors: Jian Guo, Alexandra Worden, Grant Hartzog, and Manuel Ares
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: We have available several plasmids designed for expression of Cas9, guide RNA, chloramphenicol acetyl-transferase, GFP, and beta-lactamase in Micromonas CCMP1545. For protein expression we used the promotor and 3' end elements from the endogenous RPS9 gene, and codon optimized the coding region. For expression of guide RNAs we used the Micromonas U6 snRNA promoter. DNA is available by contacting M. Ares .We have constructed and sequence verified 9 plasmids which we would like to make available to others attempting to detect transformation of DNA into Micromonas. Using the CCMP1545 genome as a source for the U6 promoter sequence, and for the promoter and 3' UTR sequences of ribosomal protein RPS9, we built the following plasmids:CRISPR/Cas9 plasmids for Micromonas1. Mp U6 promoter driving Bae cassette for guide RNA expression in pUC132. Mp RPS9-Cas9SV40-RPS9 in pUC133. Both Mp U6 promoter driving Bae cassette for guide RNA expression and Mp RPS9-Cas9SV40-RPS9 in pUC13These first three plasmids were anticipated to enable stable incorporation of transgenes at specific genomic locations. BaeI is a type IIS restriction enzyme that leaves noncompatible sticky ends. In the context of the gRNA cassette, a pair of 24 nt oligos designed to have the sticky ends compatible with BaeI cleaved plasmid are annealed and cloned into the plasmid, replacing the BaeI cassette while adding the 20 nt target complementary sequence of the desired guide RNA.Selectable/Detectable Marker genes4. Mp RPS9-codon optimized GFPsv40-RPS9 in pUC135. Mp RPS9-codon optimized chloramphenicol acetyltransferase-RPS9 in pUC13
6. Mp RPS9-codon optimized beta-lactamase-RPS9 in pUC13
These three plasmids have the indicated coding regions codon optimized for Micromonas flanked by RPS9 promoter and 3' end sequences. In the case of GFP, a nuclear localization signal from SV40 has been added to the C-terminus.
Plasmids for Agrobacterium-mediated gene transfer
7. Mp RPS9-codon optimized GFPsv40-RPS9 in pOSCAR
8. Mp RPS9-codon optimized chloramphenicol acetyltransferase-RPS9 in pOSCAR
9. Mp RPS9-codon optimized beta-lactamase-RPS9 in pOSCAR
Proper citation: Jian Guo, Alexandra Worden, Grant Hartzog, and Manuel Ares 2018. Plasmid DNAs designed for expression in Micromonas CCMP1545. protocols.io dx.doi.org/10.17504/protocols.io.i9wch7e Copy
Authors: This protocol was modified by Sabrina Geraci-Yee and is from: Pozarowski P, Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol Bio. 281:301-311.
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: Sabrina Geraci-Yee modified this protocol from: Pozarowski P., Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol.
Bio. 281:301-311.
Proper citation: This protocol was modified by Sabrina Geraci-Yee and is from: Pozarowski P, Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol Bio. 281:301-311. 2017. Staining Labyrinthulomycetes with Propidium Iodide. protocols.io dx.doi.org/10.17504/protocols.io.hfjb3kn Copy
Authors: This protocol modified from: Pandey A, Bhathena Z. 2014. Prevalence of PUFA Rich Thraustochytrids sps. along the Coast of Mumbai for Produ
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: This protocol modified from: Pandey A, Bhathena Z. 2014. Prevalence of PUFA Rich Thraustochytrids sps. along the
Coast of Mumbai for Production of Bio Oil. Journal of Food and Nutrition
Research 2(12): 993-999.
Proper citation: This protocol modified from: Pandey A, Bhathena Z. 2014. Prevalence of PUFA Rich Thraustochytrids sps. along the Coast of Mumbai for Produ 2017. Staining Unfixed Labyrinthulomycetes with Nile Red. protocols.io dx.doi.org/10.17504/protocols.io.hghb3t6 Copy
Authors: Erin Garza, Vincent Bielinski
Group: Protist Research to Optimize Tools in Genetics (PROT-G), JCVI West Protocols
Summary: This method can be used to increase the efficiency of Gibson Assemblies containing many pieces and/or difficult to assemble DNA fragments.
Proper citation: Erin Garza, Vincent Bielinski 2020. Nested Gibson Assembly. protocols.io dx.doi.org/10.17504/protocols.io.bbikikcw Copy
Can't find your Protocol?
We recommend that you click next to the search bar to check some helpful tips on searches and refine your search firstly. If you want to find a specific protocol and you know the DOI of the protocol already, it's easier to enter a DOI to search. You can refine the search results using Facets on the left side of the search results page. If you are on the table view, you can also search in a specific column by clicking the column title and enter the keywords.
If you still could not find your protocol in the search results, please help us by adding it into the system — it's easy. Create and publish your protocols at Protocols.io.
Welcome to the dkNET Resources search. From here you can search through a compilation of resources used by dkNET and see how data is organized within our community.
You are currently on the Community Resources tab looking through categories and sources that dkNET has compiled. You can navigate through those categories from here or change to a different tab to execute your search through. Each tab gives a different perspective on data.
If you have an account on dkNET then you can log in from here to get additional features in dkNET such as Collections, Saved Searches, and managing Resources.
Here is the search term that is being executed, you can type in anything you want to search for. Some tips to help searching:
You can save any searches you perform for quick access to later from here.
We recognized your search term and included synonyms and inferred terms along side your term to help get the data you are looking for.
If you are logged into dkNET you can add data records to your collections to create custom spreadsheets across multiple sources of data.
Here are the sources that were queried against in your search that you can investigate further.
Here are the categories present within dkNET that you can filter your data on
Here are the subcategories present within this category that you can filter your data on
If you have any further questions please check out our FAQs Page to ask questions and see our tutorials. Click this button to view this tutorial again.
The NIDDK Information Network (dkNET) serves the needs of basic and clinical investigators by providing seamless access to large pools of data and research resources relevant to the mission of The National Institute of Diabetes Digestive and Kidney Diseases (NIDDK). dkNET is hosted at University of California San Diego, and is supported by NIH NIDDK grant U24DK097771.
Email: info_at_ dknet.org
