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Authors: David S. Booth, Heather Middleton, and Nicole King
Group: King Lab, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This collection of protocols describes the preparation of reagents for transfecting DNA into the choanoflagellate Salpingoeca rosetta using a Nucleofector 4d Device and SF kit from Lonza. Plasmids that use S. rosetta promoters and regulatory elements to drive the expression of luciferase and fluorescent protein reporters are being deposited at Addgene. Acknowlegements: Arielle Woznica and Laura Wetzel for meticulous editing of protocol and clarifying steps in the procedure. The Gordon and Betty Moore Foundation for supporting this research.
Proper citation: David S. Booth, Heather Middleton, and Nicole King 2018. Transfection of Salpingoeca rosetta. protocols.io https://dx.doi.org/10.17504/protocols.io.h8bb9sn 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 https://dx.doi.org/10.17504/protocols.io.2atgaen Copy
Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Rachele Cesaroni 2017. Small-scale silencing experiment in vegetative Euplotes crassus (provisional). protocols.io https://dx.doi.org/10.17504/protocols.io.hi5b4g6 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 https://dx.doi.org/10.17504/protocols.io.rmud46w 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 https://dx.doi.org/10.17504/protocols.io.n83dhyn 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.
Proper citation: April Woods, G Jason Smith 2017. Modified Bacterial Conjugation Protocol For Pseudo-nitzschia multiseries. protocols.io https://dx.doi.org/10.17504/protocols.io.gj6bure Copy
Authors: Dr. Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: March, 2012; based on sample preparation guide v2, catalog # RS-930-1021, part # 15026495 Rev. A, August 2011Modifications: samples in PCR tubes (because a plate/seal will not be used, some numbered steps are skipped); include two rounds of poly-A selection (with two separate sets of beads); repeat clean-up step after PCR to remove additional adapters; elute with 1.5 ul less volume off of beads (to leave 1 ul behind, not 2.5 ul)
Proper citation: Dr. Daniel Richter 2017. TruSeq RNA Preparation with Modification for Four Rounds of Poly-A Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.imucc6w Copy
Authors: Binnypreet Kaur1, 2 , Drahomíra Faktorová1, 2, Priscila Peña-Diaz1 and Julius Lukeš1, 2
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes
Proper citation: Binnypreet Kaur1, 2 , Drahomíra Faktorová1, 2, Priscila Peña-Diaz1 and Julius Lukeš1, 2 2019. Transfection of Diplonemids using AMAXA Nucleofactor apparatus. protocols.io https://dx.doi.org/10.17504/protocols.io.hf2b3qe Copy
Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Rachele Cesaroni 2017. Quick staining procedure of nuclei in Euplotes crassus using DAPI. protocols.io https://dx.doi.org/10.17504/protocols.io.g3tbynn Copy
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: Trace metals solution to be used in ESAW Media for Marine Phytoplankton
Proper citation: Ashley Humphrey 2016. Trace Metal Solution for ESAW Media. protocols.io https://dx.doi.org/10.17504/protocols.io.gdabs2e Copy
Authors: Nick Irwin, Elisabeth Hehenberger, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Caecitellus sp. is a small phagotroph belonging to the biocosoecid order of the stramenopiles. Caecitellus is found across global oceans but it's role in marine ecosystems, as with many heterotrophic flagellates, is unclear. We isolated a strain of Caecitellus (small subunit rRNA gene was 99.53% identical to Caecitellus parvulus and Caecitellus paraparvulus) from a contaminated culture of Oxyrrhis marina and assessed its amenability to transformation. Fluorescein isothiocyanate conjugated to dextran was successfully introduced into Caecitellus sp. using a high voltage exponential decay pulse (1000 V, 10 µF, ∞ Ω) from a BioRad GenePulser Xcell (cuvette width 0.2 cm). Cell viability was confirmed by observing motility and fluorescence 24 h after electroporation (Figure 1). Lower voltage pulses did not result in FITC uptake. Estimates of transformation effifiency were difficult to obtain due to the small size of the cells.Unfortunately, the isolated strain of Caecitellus perished in an incubator malfunction. However, these results may be transferable to Caecitellus parvulus, a model for heterotrophic flagellates.
Proper citation: Nick Irwin, Elisabeth Hehenberger, Patrick Keeling 2019. Electroporation of Caecitellus sp. with FITC-dextran. protocols.io https://dx.doi.org/10.17504/protocols.io.35kgq4w Copy
Authors: Mariana Rius, Kashyapa Bandaralage
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: Sonication procedure implemented to reduce labyrinthulomycete cell aggregation from culture for subsequent growth analyses.
Proper citation: Mariana Rius, Kashyapa Bandaralage 2017. Dispersing Labyrinthulomycete Cell Aggregates by Sonication. protocols.io https://dx.doi.org/10.17504/protocols.io.hg2b3ye Copy
Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Rachele Cesaroni 2019. Long staining procedure of nuclei in Euplotes crassus using DAPI. protocols.io https://dx.doi.org/10.17504/protocols.io.2akgacw Copy
Authors: Jernej Turnsek, Chris Dupont
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Here we present an efficient way to grow Tp colonies inside a 0.25% superclean agar matrix. This protocol could possibly be applicable to other marine microeukaryotes that are problematic to grow on fully solid support.
Proper citation: Jernej Turnsek, Chris Dupont 2017. Pour plating of Thalassiosira pseudonana (Tp). protocols.io https://dx.doi.org/10.17504/protocols.io.i6qchdw Copy
Authors: Maria Rubio-Brotons
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab
Proper citation: Maria Rubio-Brotons 2017. Transient transfection of unicellular relative to animals Corallochytrium limacisporum with electroporation using Neon SystemVersion 1. protocols.io https://dx.doi.org/10.17504/protocols.io.hmwb47e Copy
Authors: Anastasios Tsaousis
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Anastasios Tsaousis 2017. Transfection of Naegleria gruberi. protocols.io https://dx.doi.org/10.17504/protocols.io.hpub5nw Copy
Authors: Philip D. Weyman
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: A sequence-independent method to turn genomic DNA into libraries of hairpins. An efficient way to get a different adapter on each end of a DNA fragment is to clone it and digest the fragment back out. Design the vector to have unique restriction sites outside the unique regions you want to put on each end. After digestion, your DNA fragments will have two different "adapters" on each end.Adapted from: Wang L., Zheng J., Luo Y., Xu T., Zhang Q., Zhang L., Xu M., Wan J., Wang MB., Zhang C., Fan Y. 2013. Construction of a genomewide RNAi mutant library in rice. Plant Biotechnology Journal 11:997–1005. DOI: 10.1111/pbi.12093.
Proper citation: Philip D. Weyman 2017. Rolling circle amplification-mediated hairpin (hp)-RNA libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.hnrb5d6 Copy
Authors: Natalia Wandyszewska and Vladimir Hampl
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: The protocol decribes the procedure and results of our attempts to transform several species of marine protists by nucleofection.
Proper citation: Natalia Wandyszewska and Vladimir Hampl 2017. Nucleofection of Pyramimonas parkeae, Chromera velia, Bigellowiella natans, Eutreptiella gymnastica, Neovalkampfia damariscottae. protocols.io https://dx.doi.org/10.17504/protocols.io.ibucanw Copy
Authors: protocols.io team
Group: VERVE Net, protocols.io news, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Protocols.io offers extensive flexibility in entering and presenting detailed methods. However, simple suggestions in here can help to make your protocol more useful to you and others. Importantly, easy steps outlined here can greatly increase the visibility of your protocol and ensure that scientists who need can find it.If you have any questions, please feel free to reach out to the protocols.io team by e-mailing us at info [at] protocols.io.
Proper citation: protocols.io team 2019. How to make your protocol more reproducible, discoverable, and user-friendly. protocols.io https://dx.doi.org/10.17504/protocols.io.g7vbzn6 Copy
Authors: Rachele Cesaroni, Angela Piersanti
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Rachele Cesaroni, Angela Piersanti 2019. Euplotes crassus micronuclear enrichment by PFGE. protocols.io https://dx.doi.org/10.17504/protocols.io.zk7f4zn Copy
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