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Authors: Ashley Humphrey
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Stock vitamin solution for ESAW Media for Marine Phytoplankton
Proper citation: Ashley Humphrey 2016. Vitamin Solution for ESAW Media for Marine Phytoplankton. protocols.io https://dx.doi.org/10.17504/protocols.io.gdcbs2w Copy
Authors: Meru J. Sadhu, Joshua S. Bloom, Laura Day, Leonid Kruglyak
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.
Proper citation: Meru J. Sadhu, Joshua S. Bloom, Laura Day, Leonid Kruglyak 2017. CRISPR-Directed Mitotic Recombination Enables Genetic Mapping Without Crosses. protocols.io https://dx.doi.org/10.17504/protocols.io.hu7b6zn Copy
Authors: G Jason Smith, April Woods
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Environmental Biotechnology Lab
Summary: Use for solid phase culture and chemical selection of Pseudo-nitzschia spp and other recalcitrant diatoms
Proper citation: G Jason Smith, April Woods 2016. Low Gel Temperature Agarose (LGTA) Media. protocols.io https://dx.doi.org/10.17504/protocols.io.gmnbu5e Copy
Authors: Aleksandra Kozyczkowska
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology
Proper citation: Aleksandra Kozyczkowska 2018. Stable tranfection of unicellular relative of animals, Corallochytrium limacisporum, using Lonza Nucleofector. protocols.io https://dx.doi.org/10.17504/protocols.io.r5ud86w Copy
Authors: Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Daniel Richter 2017. Modified Qiagen PCR purification (no gel extraction) with MinElute spin column.. protocols.io https://dx.doi.org/10.17504/protocols.io.ivece3e Copy
Authors: Lev Tsypin, Aaron Turkewitz
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes how to deposit several cells of S. robusta in droplets on a petri dish, allowing the experimenter to track the growth of approximately single cell isolates with high throughput. This was used to determine drug sensitivity.We found that in, liquid culture, S. robusta is sensitive to 10 ug/mL puromycin and 250 ug/mL glyphosate. One caveat to keep in mind is that S. robusta appears more tolerant to antibiotics when grown on agar plates, being insensitive to up to 20 ug/mL of puromycin, for example. We recommend selection in liquid culture.
Proper citation: Lev Tsypin, Aaron Turkewitz 2019. Cell deposition and drug sensitivity assay. protocols.io https://dx.doi.org/10.17504/protocols.io.336gqre Copy
Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Invitrogen's HA tag monoclonal antibody in combination with Western Breeze Anti-Mouse Chemiluminescent Western Blot Immunodetection Kit leads to no background Western blots with Pt lysates. HA tag thus represents a tag of choice for (1) efficient and unambioguous protein expression analysis, (2) immunofluorescence experiments and (3) various IP and pull-down experiments in Pt and possibly other diatoms as well as other marine microeukaryotes.
Proper citation: Jernej Turnsek 2017. HA tag enables highly efficient detection of heterologous proteins in Phaeodactylum tricornutum (Pt) exconjugants. protocols.io https://dx.doi.org/10.17504/protocols.io.j7ncrme Copy
Authors: Jeffrey Lewis, Amanda Scholes
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Lewis Lab
Summary: A generalizable protocol for measuring relative changes in gene expression via qPCR (cDNA synthesis, qPCR primer optimization, and qPCR analysis), with specific optimization for the budding yeast Saccharomyces cerevisiae.
Proper citation: Jeffrey Lewis, Amanda Scholes 2020. Quantitative (q)PCR and Differential Expression Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bbgpijvn Copy
Authors: Jacob Harris
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Alverson Lab
Summary: The FlowCam can be used to take quick cell density measurements of liquid samples.
Proper citation: Jacob Harris 2016. FlowCam Standard Operating Procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.f2tbqen Copy
Authors: Larry Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Larry Klobutcher 2017. Growth of Dunaliella salina on artificial seawater.. protocols.io https://dx.doi.org/10.17504/protocols.io.gwpbxdn Copy
Authors: Elin Einarsson
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: We have created plasmids for transfection of the parasite Perkinsus marinus using Golden Gate cloning. We have explored the possibility to either FACS sort (GFP/mCherry fused to MOE membrane protein) or using drug selection (bleomycin, blasticidin S or puromycin) to enrich transfected cells.
Proper citation: Elin Einarsson 2019. Golden Gate plasmids used for transfection of Perkinsus marinus. protocols.io https://dx.doi.org/10.17504/protocols.io.37egrje Copy
Authors: Fatma Gomaa, Virginia Edgcomb, Peter Girguis, Zhuhong li, Roberto Docampo
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This plasmid is designed to integrate within the small subunit ribosomal RNA gene region of B. saltans. It carries 1 kb of the B. saltans small subunit ribosomal RNA gene. To construct this cassette, we first sequenced the complete ribosomal operon of B. saltans, which has a total length of ̴9 kb. The structure of the ribosomal operon of B. saltans is similar in the closely related kinetoplastida Leishmania, where the ribosomal RNA genes are arranged in head- to-tail repeats (Figure 4b and Yan et al., Molecular and biochemical parasitology 103, no. 2 (1999): 197-210). For the cassette construction, we used only a 1 kb region of the 18S ribosomal RNA gene, which is represented in the schematic as Homologous Recombination Region 1 (HR1) and Homologous Recombination Region 2 (HR2) each, of which is 500 bp . The GFP and Neo genes are located in the middle of the construct and their expression is controlled by the presence of the B. saltans tubulin intergenic region (Bs TUB IGR) at the 3’ and 5’ ends of each gene. As the transcription in B. saltans is polycistronic, the presence of the tubulin intergenic region is necessary for trans-splicing and polyadenylation.
Proper citation: Fatma Gomaa, Virginia Edgcomb, Peter Girguis, Zhuhong li, Roberto Docampo 2018. Bodo saltans SSU-GFP-Neo cassette. protocols.io https://dx.doi.org/10.17504/protocols.io.s5meg46 Copy
Authors: Janina Brakel
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Proper citation: Janina Brakel 2016. Submixture for Step1 of Labyrinthula growth medium. protocols.io https://dx.doi.org/10.17504/protocols.io.ehmbb46 Copy
Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Rachele Cesaroni 2019. Small-scale silencing experiment in vegetative Euplotes crassus (provisional). protocols.io https://dx.doi.org/10.17504/protocols.io.2asgaee Copy
Authors: RACHELE CESARONI
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: RACHELE CESARONI 2017. Euplotes crassus transformation using FuGene HD Transfection Reagent as vehicle. protocols.io https://dx.doi.org/10.17504/protocols.io.g43byyn Copy
Authors: Maria Rubio-Brotons
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab
Proper citation: Maria Rubio-Brotons 2016. How to culture corallochytrean Corallochytrium limacisporum, a unicellular relative to animals. protocols.io https://dx.doi.org/10.17504/protocols.io.gqmbvu6 Copy
Authors: Andrés Ramírez, Albane Ruaud, Fernan Federici, Peter von Dassow
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol has been optimized for electroporation of natural communities in coastal surface waters. As natural communities may vary in different places we recomend to use this protocol as a starting point and re-optimize according to the results. Guidelines are provided.
Proper citation: Andrés Ramírez, Albane Ruaud, Fernan Federici, Peter von Dassow 2017. Electroporation of natural communities in sea water. protocols.io https://dx.doi.org/10.17504/protocols.io.hpnb5me Copy
Authors: Imen Lassadi
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Imen Lassadi 2017. Oyster parasite Perkinsus marinus transformation using Amaxa electroporator and non-proprietary electroporation buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.gv9bw96 Copy
Authors: Deepak Nanjappa and Kathryn Coyne
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol was developed for transformation of Heterosigma akashiwo by electroporation. This species does not grow on solid medium, so transformants are grown "in bulk" and should not be considered clonal. We have had limited success with this protocol, which may need to be further optimized.
Proper citation: Deepak Nanjappa and Kathryn Coyne 2017. Electroporation of Heterosigma akashiwo. protocols.io https://dx.doi.org/10.17504/protocols.io.hjmb4k6 Copy
Authors: April Woods, G Jason Smith
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: April Woods, G Jason Smith 2019. Modified Bacterial Conjugation Protocol For Pseudo-nitzschia multiseries. protocols.io https://dx.doi.org/10.17504/protocols.io.4pzgvp6 Copy
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