Nov 10, 2020
Version 2
Stable transfection of unicellular relative of animals, Corallochytrium limacisporum, using Lonza Nucleofector V.2
- Aleksandra Kozyczkowska1,
- Sebastián R. Najle2,
- Eduard Ocaña-Pallarès3,
- Cristina Aresté4,
- Iñaki Ruiz-Trillo3,
- Elena Casacuberta5
- 1Institut de Biologia Evolutiva (UPF-CSIC) Barcelona;
- 2Center for Genomic Regulation (CRG);
- 3Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra);
- 4IIBB;
- 5Institute of Evolutionary Biology
- Protist Research to Optimize Tools in Genetics (PROT-G)
- Multicellgenomelab
Protocol Citation: Aleksandra Kozyczkowska, Sebastián R. Najle, Eduard Ocaña-Pallarès, Cristina Aresté, Iñaki Ruiz-Trillo, Elena Casacuberta 2020. Stable transfection of unicellular relative of animals, Corallochytrium limacisporum, using Lonza Nucleofector. protocols.io https://dx.doi.org/10.17504/protocols.io.bpfgmjjwVersion created by Aleksandra Kozyczkowska
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: November 05, 2020
Last Modified: November 10, 2020
Protocol Integer ID: 44232
Keywords: transfection, electroporation, unicellular organism, stable
Abstract
This protocol describes the transfection of Corallochytrium limacisporum, one of the unicellular relatives of animals. The transfection is performed using Lonza Nucleofector System X and reporter cassettes that contain endogenous regulatory elements of C. limacisporum.
Guidelines
Keeps cells on ice during the whole procedure & all reagents must be ice-cold
Materials
Main materials:
P3 PrimaryCell 4D-Nucleofector Kit S (32 rxn) Kit Catalog# H3V4XP-5032
pUC19 carrier plasmid
highly concentrated reporter construct
1XPBS
Marine Broth
Cell Culture preparation
Cell Culture preparation
Inoculate 100 μl of C. limacisporum culture in 5ml of Marine Broth in 25cm2 flask. Keep at 23°C air incubator for two days.
Cell count
Cell count
Scrape gently and count cells to reach 1.5E6 cells / condition using hemocytometer
If there are more conditions, it is recommended to pull the cells together to get more visible pellet
Medium removal
Medium removal
Spin down the cells at 1500xg for 5'
Discard the supernatant.
Washing step
Washing step
5m
5m
Wash the pellet with an ice-cold 1XPBS (100 μl / condition)
Spin down and discard the supernatant.
Prepare DNA mix
Prepare DNA mix
Mix DNA plasmids: 10 μg of an reporter plasmid + 20 μg carrier DNA (pUC19)
It is recommended that plasmid is highly concentrated (2-5 μg/ul) to avoid final buffer dilution with the DNA solution
Our plasmid contains mCherry coding sequence & resistance to puromycin gene (pac) under constitutive promoter: actin (Addgene: #104446) or tubulin (Addgene#129560)
Note: 10 μg of reporter plasmids guarantees the highest efficiency, however a successful transfection can be also obtained with 5 and 1μg of the plasmid
Transfer DNA mix to 20 μl of ice-cold P3 buffer
It is recommended not to keep the cells in P3 buffer for too long
Add DNA mix to the cell pellet
Transfer the mix (cells + DNA plasmid) into a strip (from the manufacture kit)
Do it carefully & without creating bubbles
Final volume: 20-25 μl
Transfection of plasmid DNA into C. limacisporum
Transfection of plasmid DNA into C. limacisporum
Insert into Lonza nucelofection system and apply code: EN - 138
Recovery step
Recovery step
After transfection, immediately add 80 μl of Marine Broth medium (MB) to a well
Mix up and down
Transfer to 12-well plate (NUNC) that contains 1 ml of growth medium (MB) and incubate overnight
Check-out for results
Check-out for results
Check for positive cells 24 - 48h post-transfection using fluorescent microscopy
Enrichment & selection of transfected cells
Enrichment & selection of transfected cells
Replace 500 μl of medium with fresh MB that contains 300 μg/ml of puromycin
Increase of mCherry-positive cells can be observed over time.