Protocols   Select Another Resource Report Type

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

Proper citation: David Booth 2018. Protocol B: Preparation of ATP for transfection. protocols.io https://dx.doi.org/10.17504/protocols.io.h8ub9ww Copy   

•   Source:

Authors: Fatma Gomma, Paulo A. Garcia, Jennifer Delaney, Peter R. Girguis, Cullen R. Buie, Virginia P. Edgcomb
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: The process of developing transient transfection protocols in P. caudatus involved initial testing and determination of proper electroporation buffers and parameters (voltage strength, pulse duration and number) to maximize cell viability. Our results suggest that the type of electroporation buffer is critical for maintaining cell viability throughout the experiment and is essential for determining the optimum electric field range.

Proper citation: Fatma Gomma, Paulo A. Garcia, Jennifer Delaney, Peter R. Girguis, Cullen R. Buie, Virginia P. Edgcomb 2017. Parabodo caudatus post-electroporation cell viability quantification using exponential decay electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.hmhb436 Copy   

•   Source:

Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Rachele Cesaroni 2019. Euplotes crassus transfection through microinjection into the macronucleus. protocols.io https://dx.doi.org/10.17504/protocols.io.2argad6 Copy   

•   Source:

Authors: Steven Wilhelm
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), CyanoHABs
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.Adapted from Nanodrop ND-1000 User Manual 

Proper citation: Steven Wilhelm 2017. Nanodrop Spectrophotometer (ND-1000) for Nucleic Acid. protocols.io https://dx.doi.org/10.17504/protocols.io.id2ca8e Copy   

•   Source:

Authors: Alle Lie
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology

Proper citation: Alle Lie 2016. The culturing of axenic Ochromonas sp. strain CCMP1393. protocols.io https://dx.doi.org/10.17504/protocols.io.ey8bfzw Copy   

•   Source:

Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Map and nucleotide sequence of the conjugation episome used to generate a TpSIl3p-EGFP expressing T. pseudonana strain reported in Faktorová D. et al.

Proper citation: Jernej Turnsek 2019. pTpPuc3_TpSil3p-eGFP. protocols.io https://dx.doi.org/10.17504/protocols.io.7ghhjt6 Copy   

•   Source:

Authors: This protocol is modified from: Raghukumar S, Schaumann K. 1993. An epifluorescence microscopy method for direct detection and enumeration of the, the thraustochytrids. Limnol. Oceanogr.
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: This protocol is modified from: Raghukumar S, Schaumann K. 1993. An epifluorescence microscopy method for direct detection and enumeration of the fungilike marine protists, the thraustochytrids. Limnol. Oceanogr. 38(1): 182-187.

Proper citation: This protocol is modified from: Raghukumar S, Schaumann K. 1993. An epifluorescence microscopy method for direct detection and enumeration of the, the thraustochytrids. Limnol. Oceanogr. 2017. Acriflavine Direct Detection Technique for Labyrinthulomycetes. protocols.io https://dx.doi.org/10.17504/protocols.io.hfib3ke Copy   

•   Source:

Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Rachele Cesaroni 2019. Quick staining procedure of nuclei in Euplotes using DAPI. protocols.io https://dx.doi.org/10.17504/protocols.io.2ajgacn Copy   

•   Source:

Authors: Anna Vanclová and Naoji Yubuki
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Organisms used for DNA transformation experiments were tested for sensitivity to five antibiotics in two different concentrations. Cell culture growing in F2 media with 3% seawater was divided to fifteen 15ml tubes, i.e. three tubes for testing each of the antibiotics: antibiotic was added to two samples to the final concentrations noted in Table 1, the third tube in the set served as a control. The cultures were incubated under 12h light cycle and 20°C and checked by light microscophy 1, 4, 7 and 19 days after the addition of antibiotics. ABCDEF1antibiotic puromycin blasticidin geneticin hygromycin zeocin 2concentration 1 5 μg/ml 5 μg/ml 200 μg/ml 200 μg/ml 100 μg/ml 3concentration 2 10 μg/ml 10 μg/ml 400 μg/ml 400 μg/ml 200 μg/ml Table 1: Tested antibiotics and concentrations

Proper citation: Anna Vanclová and Naoji Yubuki 2017. Antibiotic sensitivity testing - Pyramimonas, Bigelowiella, Eutreptiella, Neovahlkampfia, Pseudonitzschia. protocols.io https://dx.doi.org/10.17504/protocols.io.hh3b38n Copy   

•   Source:

Authors: Paulo A. Garcia, Zhifei Ge, Laura Kelley, Steven Holcomb, Cullen R. Buie
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Synthetic biology holds great potential for addressing pressing challenges for mankind and our planet. One technical challenge in tapping into the full potential of synthetic biology is the low efficiency and low throughput of genetic transformation for many types of cells. In this paper, we discuss a novel microfluidic system for improving bacterial electrotransformation efficiency and throughput. Our microfluidic system is comprised of non-uniform constrictions in microchannels to facilitate high electric fields with relatively small applied voltages to induce electroporation. Additionally, the microfluidic device has regions of low electric field to assist in electrophoretic transport of nucleic acids into the cells. The device features hydro- dynamically controlled electric fields that allow cells to experience a time dependent electric field that is otherwise difficult to achieve using standard electronics. Results suggest that transformation efficiency can be increased by ∼4×, while throughput can increase by 100–1000× compared to traditional electroporation cuvettes. This work will enable high-throughput and high efficiency genetic transformation of microbes, fa- cilitating accelerated development of genetically engineered organisms.

Proper citation: Paulo A. Garcia, Zhifei Ge, Laura Kelley, Steven Holcomb, Cullen R. Buie 2017. High Efficiency Microfluidic Electrotransformation. protocols.io https://dx.doi.org/10.17504/protocols.io.hapb2dn Copy   

•   Source:

Authors: Yoshihisa Hirakawa, Hirokazu Sakamoto, Takashi Shiratori, Elisabeth Hehenberger, Nick Irwin, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Yoshihisa Hirakawa, Hirokazu Sakamoto, Takashi Shiratori, Elisabeth Hehenberger, Nick Irwin, Patrick Keeling 2018. Antibiotic sensitivity test for dinoflagellates and Chromera. protocols.io https://dx.doi.org/10.17504/protocols.io.vcke2uw Copy   

•   Source:

Authors: Senjie Lin, Huan Zhang, Brittany Sprecher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol is intended to work for many dinoflagellates but focusses on the method used to successfully transform Karlodinium veneficum.

Proper citation: Senjie Lin, Huan Zhang, Brittany Sprecher 2019. Nucleofector Protocol for Dinoflagellates using Lonza’s 4D-Nucleofector X Unit. protocols.io https://dx.doi.org/10.17504/protocols.io.7n8hmhw Copy   

•   Source:

Authors: Inbal Nussbaum, Daniella Schatz
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: A protocol for generating single colonies of Emiliania huxleyi embedded in soft-agarose. 

Proper citation: Inbal Nussbaum, Daniella Schatz 2018. Pour plating protocol for Emiliania huxleyi (single colonies). protocols.io https://dx.doi.org/10.17504/protocols.io.p4ydqxw Copy   

•   Source:

Authors: Claudio Slamovits
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Slamovits Lab
Summary: !!! ~~~~~ IN DEVELOPMENT ~~~~~ !!!This approach uses in-vitro transcription to generate a dinoflagellate-type mRNA (i.e. capped with the dino-SL leader), which is introduced into cells using an RNA transformation reagent. Screening for expression of the protein encoded in the mRNA follows.

Proper citation: Claudio Slamovits 2017. mRNA approach for transient expression in dinoflagellates. protocols.io https://dx.doi.org/10.17504/protocols.io.gwgbxbw Copy   

•   Source:

Authors: Yoshihisa Hirakawa, Hirokazu Sakamoto, Elisabeth Hehenberger, Nick Irwin, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Yoshihisa Hirakawa, Hirokazu Sakamoto, Elisabeth Hehenberger, Nick Irwin, Patrick Keeling 2018. In vitro synthesized mRNA transfection to Perkinsus marinus. protocols.io https://dx.doi.org/10.17504/protocols.io.vcme2u6 Copy   

•   Source:

Authors: David S. Booth, Heather Middleton, and Nicole King
Group: King Lab, Protist Research to Optimize Tools in Genetics (PROT-G), Alegado Lab
Summary: The protocol describes the transformation of 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. 

Proper citation: David S. Booth, Heather Middleton, and Nicole King 2018. Protocol I: Transfection of Salpingoeca rosetta with Lonza 4d nucleofector. protocols.io https://dx.doi.org/10.17504/protocols.io.h68b9hw Copy   

•   Source:

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)

Proper citation: Ashley Humphrey 2016. ESAW Media for Marine Phytoplankton. protocols.io https://dx.doi.org/10.17504/protocols.io.gc9bsz6 Copy   

•   Source:

Authors: Laura Halligan, Jackie Collier
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: This protocol is a two-part lipid extraction for labyrinthulomycetes. The only strain we have tried this protocol on so far is Aurantiochytrium limacinum ATCC MYA-1381.This protocol was modified from: Higgins, B.T., A. Thornton-Dunwoody, J.M. Labavitch, J.S. VanderGheynst. 2014. Microplate assay for quantitation of neutral lipids in extracts from microalgae. Anal. Biochem. 465: 81-89. 

Proper citation: Laura Halligan, Jackie Collier 2018. Lipid extractions from labyrinthulomycetes . protocols.io https://dx.doi.org/10.17504/protocols.io.qbddsi6 Copy   

•   Source:

Authors: Lev Tsypin, Aaron Turkewitz
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes the culturing conditions used to grow S. robusta in the lab for all experiments.

Proper citation: Lev Tsypin, Aaron Turkewitz 2019. Media and reagents for Seminavis robusta cultivation and experiments. protocols.io https://dx.doi.org/10.17504/protocols.io.33xgqpn Copy   

•   Source:

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.zwff7bn Copy   

•   Source: