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Authors: Imen Lassadi
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Attempts to transfect Hematodinium was performed using a plasmid that will express eGFP under the control of DVNP regulatory sequences. See attachment for plasmid information.
Proper citation: Imen Lassadi 2019. Plasmid used for transfection trails of Hematodinium . protocols.io https://dx.doi.org/10.17504/protocols.io.4nigvce Copy
Authors: Vincent Bielinski, Chris Dupont
Group: Protist Research to Optimize Tools in Genetics (PROT-G), JCVI West Protocols
Summary: This protocol is the adaptation of previously published methods for use in isolating intact high molecular weight DNA from stramenopiles for long-read sequencing. The advantages here are minimal amount of centrifugation steps on isolated DNA, as well as the long-term storage of the DNA while in plugs to allow experimental flexibility.
Proper citation: Vincent Bielinski, Chris Dupont 2018. Modified HMW DNA Isolation from Stramenopiles with Agar Plugs. protocols.io https://dx.doi.org/10.17504/protocols.io.wenfbde Copy
Authors: Lu Wang, Brittany Sprecher, Huan Zhang, and Senjie Lin
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Lu Wang, Brittany Sprecher, Huan Zhang, and Senjie Lin 2019. Co-Incubation protocol for transforming heterotrophic dinoflagellates (e.g. Oxyrrhis marina). protocols.io https://dx.doi.org/10.17504/protocols.io.7pphmmn Copy
Authors: Gina Filloramo
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Successful transformation of Phaeodactylum tricornutum (CMP632) by electroporation was achieved based on a modified version of Zhang & Hu (2014).
Proper citation: Gina Filloramo 2018. Transformation of Phaeodactylum tricornutum by electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.s4cegsw Copy
Authors: RACHELE CESARONI, Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: RACHELE CESARONI, Rachele Cesaroni 2019. Euplotes crassus transfection using Lipofectamine 2000 as vehicle (provisional). protocols.io https://dx.doi.org/10.17504/protocols.io.2aigace Copy
Authors: Glen Wheeler, Andrea Highfield, Rowena Stern
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: pIgNAT vectorcontains nourseothricin-resistance gene (NAT) codon-optimised for expression in Isochrysis galbana, flanked by promoter/terminator from E. huxleyi HSP70.
Proper citation: Glen Wheeler, Andrea Highfield, Rowena Stern 2019. pIgNAT vector. protocols.io https://dx.doi.org/10.17504/protocols.io.8frhtm6 Copy
Authors: Joshua Bugge, Deborah Robertson
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Robertson Lab
Summary: An electroporation-mediated genetic transformation or the marine diatom Thalassiosira pseudonana was developed. Using a sorbitol-based buffer, T. pseudonana cells were successfully transformed with the Tpfcp/nat plasmid at an efficiency of 2820 per 108 cells. This represents a six-fold improvement compared with previously published methods.
Proper citation: Joshua Bugge, Deborah Robertson 2019. Electroporation of Thalassiosira pseudonana. protocols.io https://dx.doi.org/10.17504/protocols.io.yvqfw5w Copy
Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Rachele Cesaroni 2017. Long staining procedure of nuclei in Euplotes crassus using DAPI. protocols.io https://dx.doi.org/10.17504/protocols.io.hjgb4jw 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 https://dx.doi.org/10.17504/protocols.io.iskcecw Copy
Authors: Inbal nussbaum, Daniella Schatz
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: We used the NEPA electroportor to transform Emiliania huxleyi cells. We were able to establish that this method can be used to introduce proteins into the cells.
Proper citation: Inbal nussbaum, Daniella Schatz 2018. NEPA electroporation of Emiliania huxleyi cells. protocols.io https://dx.doi.org/10.17504/protocols.io.p4zdqx6 Copy
Authors: Steven Wilhelm, Robbie Martin
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), CyanoHABs, Great Lakes Center for Fresh Waters and Human Health
Summary: Contact Dr. Steven Wilhelm ([email protected]) or Robbie Martin ([email protected]) for additional information regarding this protocol.
Proper citation: Steven Wilhelm, Robbie Martin 2017. Hot-phenol RNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.g89bzz6 Copy
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 https://dx.doi.org/10.17504/protocols.io.jy7cpzn Copy
Authors: G Jason Smith
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Environmental Biotechnology Lab
Summary: This is a modification of the origianl Chelex 100 extraction described by Walsh, Metzger and Higuchi (1991 BioTechniques 10(4):506). Adding PVPP facilitates working with environmental samples as well as pure cultures hi in phenolics or other contaminants.
Proper citation: G Jason Smith 2017. Simple DNA Extraction for Phytoplankton Using Chelex 100. protocols.io https://dx.doi.org/10.17504/protocols.io.hinb4de Copy
Authors: RACHELE CESARONI, Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: RACHELE CESARONI, Rachele Cesaroni 2019. Dot blot to verify microinjection efficacy in Euplotes crassus. protocols.io https://dx.doi.org/10.17504/protocols.io.2aqgadw Copy
Authors: Fatma Gomaa, Roberto Docampo, Zhuhong Li, Peter Girguis, Virginia Edgcomb
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Stable transfection of B. saltans has been achieved using plasmid for EF 1 alpha C terminal tagging.
Proper citation: Fatma Gomaa, Roberto Docampo, Zhuhong Li, Peter Girguis, Virginia Edgcomb 2019. Bodo saltans transfection with plasmid for tagging EF1alpha gene__IG BsTub. protocols.io https://dx.doi.org/10.17504/protocols.io.7fchjiw Copy
Authors: Lawrence A. Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Numerous unpublished studies indicate that Euplotes crassus is resistant to extremely high concentrations of commonly used selective agents when grow in artificial seawater. We have found that when E. crassus is grown in 1 part artificial seawater + 9 parts 0.3 M glucose, they are much more sensitive to a number of selective agents. For example, 1080 ug/ml paromomycin in artificial seawater only inhibited growth, as little as 120 ug/ml was effective in killing cells when grown with 0.3 M glucose. Similarly, G418 at 400 ug/ml had little effect in seawater, but as little as 100 ug/ml blocked growth in 0.3 M glucose. This may not prove true for all selective agents, as we saw no effect with paclitaxel up to 100 uM under either growth condition.
Proper citation: Lawrence A. Klobutcher 2017. Increased sensitivity of Euplotes crassus to selective agents using 0.3 M glucose-based culture conditions.. protocols.io https://dx.doi.org/10.17504/protocols.io.gwnbxde Copy
Authors: Erin Garza, Vincent Bielinski
Group: Protist Research to Optimize Tools in Genetics (PROT-G), JCVI West Protocols
Summary: A high-throughput method for measuring β-glucuronidase (GUS) activity in the diatom Phaeodactylum tricornutum. This protocol has been optimized for 250 μl volumes. For larger volumes see the following protocol dx.doi.org/10.17504/protocols.io.hefb3bn, which this protocol was based off of.
Proper citation: Erin Garza, Vincent Bielinski 2020. High-Throughput Beta-glucuronidase (GUS) assay for Phaeodactylum tricornutum. protocols.io https://dx.doi.org/10.17504/protocols.io.bbexijfn Copy
Authors: Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Daniel Richter, Nov 29, 2011based on RNAqueous May 29, 2008 protocol revision C, TURBO DNA-free June 9, 2009 protocol 1907M revision F
Proper citation: Daniel Richter 2017. RNAqueous with TURBO DNA-free and SUPERase-In. protocols.io https://dx.doi.org/10.17504/protocols.io.iqzcdx6 Copy
Authors: Janina Brakel
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Proper citation: Janina Brakel 2016. Labyrinthula growth medium. protocols.io https://dx.doi.org/10.17504/protocols.io.ehkbb4w Copy
Authors: Lawrence A. Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: The electroporation procedure and conditions described allow for the entry of DAPI into the cell, resulting in nuclear staining. We have not yet been successful in using these conditions for transformation of Euplotes crassus, but only a couple of constructs have been tried to date.
Proper citation: Lawrence A. Klobutcher 2017. Electroporation of Euplotes crassus: conditions that allow for the uptake of DAPI.. protocols.io https://dx.doi.org/10.17504/protocols.io.gwmbxc6 Copy
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