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Authors: Tamara Matute, Isaac Nuñez, Fernan Federici, Peter von Dassow
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Laboratorio de Tecnologias Libres
Summary: Plasmid maps and genbank sequence files for the plasmids used in the transformation of Nannochloropsis oceanica and environmental samples following the protocol Matute et al..

Proper citation: Tamara Matute, Isaac Nuñez, Fernan Federici, Peter von Dassow 2019. Plasmid maps and genbank files. protocols.io https://dx.doi.org/10.17504/protocols.io.7nrhmd6 Copy   

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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: Defined Medium for Aureococcus anophagefferens

Proper citation: Ashley Humphrey 2017. ASP12A Recipe for culturing Aureococcus anophagefferens. protocols.io https://dx.doi.org/10.17504/protocols.io.g29byh6 Copy   

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Authors: Senjie Lin
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Protocol of dinoflagellate cell transformation

Proper citation: Senjie Lin 2016. Dinoflagellate transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.e6bbhan Copy   

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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 https://dx.doi.org/10.17504/protocols.io.e5nbg5e Copy   

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Authors: Dr. Steven Wilhelm, Samantha Coy
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Samantha R Coy's Protocols
Summary: Van Etten, J. (n.d.). Formulation of Modified Bold's Basal Medium (MBBM). Retrieved from http://ncv.unl.edu/vanettenlab/ Contact Dr. Steven Wilhelm ([email protected]) or Samantha Coy ([email protected]) for additional information regarding this protocol.

Proper citation: Dr. Steven Wilhelm, Samantha Coy 2017. MBBM Media. protocols.io https://dx.doi.org/10.17504/protocols.io.hg3b3yn Copy   

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Authors: Adrian Clarke
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Adrian Clarke 2017. Axenic treatment of Skeletonema Marinoi for DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.g6cbzaw Copy   

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Authors: Monica Berjon-Otero, Sarah Duponchel, Matthias Fischer
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Fluorescence microscopy is essential to understand, between others, the cell organization and the life cycle of viruses that infect them. This protocol describes the steps neccesary to fix and permeabilize Cafeteria roenbergensis cells allowing its staining with antibodies or chemical reactions as Click-IT reactions.

Proper citation: Monica Berjon-Otero, Sarah Duponchel, Matthias Fischer 2019. Fluorescence microscopy with the marine heterotrophic flagellate Cafeteria roenbergensis. protocols.io https://dx.doi.org/10.17504/protocols.io.qjmduk6 Copy   

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Authors: Joshua Rest, Jackie Collier
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: We analyzed the recently available whole genome sequences from two thraustochytrids (Aurantiochytrium limacinum ATCC MYA-1381, Schizochytrium aggregatum ATCC 28209) and one aplanochytrid (Aplanochytrium PBS07) We then calculated the genome-wide relative synonymous codon usage, codon frequencies and GC content for predicted coding sequences from each of the three species. We compared these to other stramenopiles: the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, and the oomycete Phytophthora sojae, as well as to the ascomycete fungus Saccharomyces cerevisiae. See this page for further description.This code was run in R version 3.3.2 (2016-10-31)Package info: RCurl_1.95-4.8 bitops_1.0-6   ape_4.0        reshape_0.8.6  seqinr_3.3-3

Proper citation: Joshua Rest, Jackie Collier 2017. Labyrinthulomycete genome codon usage calculation code. protocols.io https://dx.doi.org/10.17504/protocols.io.hgyb3xw Copy   

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Authors: David Booth
Group: King Lab, Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: David Booth 2018. Protocol E: Preparation of pretreatment buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.h8xb9xn Copy   

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Authors: Eric Gann
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Defined Medium for Aureococcus anophagefferens

Proper citation: Eric Gann 2016. ASP12A Recipe for culturing Aureococcus anophagefferens. protocols.io https://dx.doi.org/10.17504/protocols.io.f3ybqpw Copy   

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Authors: Alyssa Alsante
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Van Etten, J. (n.d.). Formulation of Modified Bold's Basal Medium (MBBM). Retrieved from http://ncv.unl.edu/vanettenlab/

Proper citation: Alyssa Alsante 2016. MBBM Stock Solutions. protocols.io https://dx.doi.org/10.17504/protocols.io.geqbtdw Copy   

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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.g3ubynw Copy   

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Authors: Elena Casacuberta, Cristina Aresté
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology
Summary: Minimal protocol for testing selective agents for Abeoforma whisleri, Sphaeroforma arctica, Pirum gemmata

Proper citation: Elena Casacuberta, Cristina Aresté 2019. Testing selective agents for the Icthyosporeans Abeoforma whisleri, Pirum gemata and Spaheroforma artica. protocols.io https://dx.doi.org/10.17504/protocols.io.z5nf85e Copy   

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Authors: Mariana Rius
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: This protocol results in the isolation of single nucleated zoospores from a culture of Aurantiochytrium limacinum. The point is to recover single nucleus colonies rather than single cell colonies (which may contain varying nuclei - particularly following transformation).

Proper citation: Mariana Rius 2018. Zoospore Isolation of Aurantiochytrium limacinum. protocols.io https://dx.doi.org/10.17504/protocols.io.u73ezqn Copy   

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Authors: Angela Piersanti
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Angela Piersanti 2019. Biolistic Transfection in Euplotes crassus (provisional). protocols.io https://dx.doi.org/10.17504/protocols.io.zk6f4ze Copy   

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Authors: Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Phenol: chloroform extraction to clean up RNA ( e.g. to remove DNAse or DNAse Inactivation Reagent.) Based on 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: Daniel Richter 2017. RNA clean-up by phenol:chloroform. protocols.io https://dx.doi.org/10.17504/protocols.io.iv4ce8w Copy   

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Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This revisited Thalassiosira pseudonana conjugation protocol builds on the following two protocols: Conjugation of Thalassiosira pseudonana (DOI: dx.doi.org/10.17504/protocols.io.f55bq86) & Pour plating of Thalassiosira pseudonana (Tp) (DOI: dx.doi.org/10.17504/protocols.io.jfncjme). Readers are strongly encouraged to read both before using this one. Gene-EGFP fusion depicted in the 'Expected Results' section was expressed under consitutive Tp-derived fcp promoter and terminator using pTpPuc3 episome backbone described in Karas et al. (2015). 

Proper citation: Jernej Turnsek 2018. Revisited Thalassiosira pseudonana (Tp) conjugation protocol enables fusion protein delivery to Tp frustule. protocols.io https://dx.doi.org/10.17504/protocols.io.nbzdap6 Copy   

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Authors: Sarah Hu
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology
Summary: This protocol describes the extraction process of RNA and/or DNA from marine environmental samples. Samples are assumed to be collected via filtering sample seawater onto filters. Filters are then flash frozen in the first lysis buffer. The protocol describes the steps used to lyse and separate material from filters and use the lysate for RNA and/or DNA extraction using Qiagen extraction kits (e.g. https://www.qiagen.com/us/shop/Sample-Technologies/Combined-Sample-Technologies/). Downstream RNA and DNA is used for molecular analyses such as tag sequencing (16S or 18S), (meta)genomic, or (meta)transcriptomic applications. List of materials needed before starting is in Guidelines.

Proper citation: Sarah Hu 2016. RNA (and optional DNA) extraction from environmental samples (filters). protocols.io https://dx.doi.org/10.17504/protocols.io.ewnbfde Copy   

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Authors: Angela Piersanti
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Angela Piersanti 2019. Transfection by Electroporation in Euplotes crassus. protocols.io https://dx.doi.org/10.17504/protocols.io.xjgfkjw Copy   

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Authors: Bernardo Pollak
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol is used for performing Type IIS assembly by either BsaI or SapI-mediated restriction/ligation using Loop assembly with either Loop or uLoop plasmids.Loop assembly comprises 8 receiver plasmids in odd and even levels (4 per level), which contain directional overhangs for higher level assembly. Odd and even level receiver plasmids are used in a recursive schema for assembly in iterative 'loops'. Any assembly (except for L0 part composition into a L1 transcriptional unit) requires the usage of 4 donor plasmids (each in a specific position) and one receiver plasmid. BsaI is used for assembly into odd levels using kanamycin selection, and SapI is used in even levels using spectynomycin selection. Loop odd receivers use the Common Syntax standard for L0 part assembly into TUs (Patron, et al. 2015 [10.1111/nph.13532]). Then, 4 L1 plasmids (positions 1-4) are assembled into an even receiver plasmid to provide a L2 assembly (4 TUs). For higher level assemblies the same assembly structure is followed, 4 L2 plasmids (positions 1-4) with an odd receiver to yield a L3 assembly (16 TUs).The Loop type IIS assembly protocol is based on: “Patron, NJ (2016) DNA Assembly for Plant Biology. Current Protocols in Plant Biology 1:1-13 [doi: 10.1002/cppb.20038]”, but with slight modifications for DNA concentrations:This protocol uses a target amount of each donor DNA of 15 fmol and 7.5 fmol for receiver plasmid DNA in a 10 µL reaction. In order to perform accurate dispensing, the plasmid DNA is diluted to their corresponding concentration of 15 fmol/µL for donor parts and of 7.5 fmol/µL for the receiver plasmid, and then 1 µL of each plasmid is added to the DNA mix. The plasmid part target concentration (in ng/uL) equals to each donor plasmid length / 100, and of the receiver plasmid length / 200. Volumes CAN be reduced by half, but expect more variability from aliquoting.

Proper citation: Bernardo Pollak 2019. Loop and uLoop assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.yxnfxme Copy   

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