May 13, 2018
Transfection of Capsaspora owczarzaki using calcium-phosphate precipitation
- Helena Parra Acero1,
- Núria Ros2,
- Alberto Perez-Posada2,
- Aleksandra Kozyczkowska2,
- Sebastián R. Najle2
- 1IBE UPF-CSIC;
- 2Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra)
External link: https://doi.org/10.1242/dev.162107
Protocol Citation: Helena Parra Acero, Núria Ros, Alberto Perez-Posada, Aleksandra Kozyczkowska, Sebastián R. Najle 2018. Transfection of Capsaspora owczarzaki using calcium-phosphate precipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.p4adqse
Manuscript citation:
Parra-Acero H, Ros-Rocher N, Perez-Posada A, Kożyczkowska A, Sánchez-Pons N, Nakata A, Suga H, Najle SR, Ruiz-Trillo I, Transfection of , a close unicellular relative of animals. Development (Cambridge, England) 145(10). doi: 10.1242/dev.162107
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
We use this protocol and it's working
Created: May 13, 2018
Last Modified: May 13, 2018
Protocol Integer ID: 12130
Keywords: Capsaspora, unicellular eukaryote, transfection
Abstract
This is a protocol to transiently transfect Capsaspora owczarzaki, a unicellular eukaryote
Guidelines
-This protocol is set up for transfecting Capsaspora in 12 wells/plate. All volumes are defined per well.
-Prepare reagents in advance
-When replicates are to be done, a larger DNA mix can be set up
Safety warnings
- Work in a laminar flow hood to ensure the experiment is not contaminated
-There are certain critical steps in this protocol that affect efficiency:
CRITICAL STEP 1: Adherent stage cells in confluency. Cultures should be fresh to maximize transfection efficiency. Ideally, they should be maintained weekly, and used for transfection at their exponential growth phase. Do not let cultures reach higher cell densities (<5x107 cells mL -1 ).
CRITICAL STEP 2: DNA-Calcium- phosphate precipitates formation. Check the cultures periodically under the microscope to check crystal size. Big cloudy precipitates may compromise
transfection efficiency. Instead, verify that small grains of refractant material are spread homogeneously in the plate.
CRITICAL STEP 3: Glycerol shock. Incubation with glycerol at this concentration should not exceed 1 min, counting from the first droplet, to avoid excessive cell death
Before start
Transfection Reagents preparation
Growth medium (for 1 L): 10 g Peptone (BD, #211677), 10 g Yeast Extract (BD, #212750), 1 g
Yeast nucleic acid (Ribonucleic Acid, Type VI from Torula Yeast) (Sigma, #R-6625), 15 mg Folic acid
(Sigma, #F8758) in 880 mL distilled water. Autoclave for 15 min at 121ºC. Cool down and aseptically
add 0.4 mL of Hemin stock solution* (Sigma, #H9039), 20 mL Buffer solution** and 100 mL of heat-
inactivated Fetal Bovine Serum (Sigma, #F9665-100ml). Filter-sterilise through 0.22 µm and store at
4ºC.
*Hemin stock solution (for 200 mL): 400 mg NaOH in 200 mL dH 2 O. Add 500 mg of Hemin and
autoclave 20 min at 121ºC. Store at 4ºC protected from the light.
**Buffer solution (for 1 L): 18.1 g KH 2 PO 4 (Sigma, #P5655), 25 g Na 2 HPO 4 (Sigma, #S5136) in 1 L
distilled water. Adjust final pH to 6.5 with HCl 37% and filter-sterilise through 0.22 µm. Store at 4ºC.
Transfection medium (for 1 L): 10 g Peptone, 15 mg Folic Acid in 990 mL distilled water. Autoclave
for 20 min at 121ºC. Aseptically add 10 mL HEPES 1 M (Sigma, #H4034) to a final concentration of
10 mM and 2.1 g Bis-Tris methane (Sigma, #B9754) final concentration 0.21% w/w. adjust pH to 7.1
with NaOH, filter-sterilise through 0.22 µm and store at 4ºC.
2X HBS (for 250 mL): Dissolve 4 g NaCl (Sigma, #S3014), 0.18 g KCl (Sigma, #P9541), 0.05 g
Na 2 HPO 4 (Sigma, #S5136), 2.5 g HEPES and 0.5 g D-glucose (Sigma, #G8270) in autoclaved distilled
water. Adjust pH to 7.1 with NaOH. Filter-sterilise through 0.22 µm, flash-freeze with liquid Nitrogen
and store at -80ºC.
1.25M CaCl 2 (for 10 mL): 1.84 g CaCl 2 (Sigma, #C1016) in 10 mL autoclaved distilled water. Filter-
sterilise through 0.22 µm, flash-freeze with liquid Nitrogen and store at -80ºC.
10% glycerol (for 4 mL): 0.8 mL of filter-sterilised 50% (v/v) glycerol (Sigma, #G7757) in 1.2 mL
autoclaved distilled water and 2 mL 2X HBS. Filter-sterilise through 0.22 µm, flash-freeze with liquid
Nitrogen and store at -80ºC.
Preparation of cells
Preparation of cells
Seed 107 cells in a flask with 5mL of growth medium, let grow for 24h at 23ºC to have a conflluent culture.
Take the cells from the confluent culture, seed 2*106 cells per well in 600 μL of growth medium.
Calcium-phosphate precipitation
Calcium-phosphate precipitation
Replace growth medium with 600 μL of transfection medium, incubate 30 min at 18ºC (room temperature).
While incubating with transfection medium, prepare DNA mix (300 μLper experiment):
1.271 pmol of plasmid in distilled water (add water to reach a total of 120 μL)
150 ul of 2X HBS (to obtain final concentration 1X HBS)
30ul of 1.25 M CaCl2 (add dropwise and while flickering the tube, to obtain a final concentration of 125mM, then invert twice to ensure mixing)
Incubate 10 min at 37ºC.
Remove medium and add 300 μL of DNA mix dropwise in the center of the wells, incubate 30min at 18ºC (room temperature).
Add 500 μL of transfection medium, incubate at least 4h at 23ºC.
Remove medium and perform an osmotic shock with 110 μL of 10% glycerol shock for 1 min.
Remove glycerol and add 600 μL of growth medium.
Put cells back at 23ºC until screening.
Screening of positive cells
Screening of positive cells
If the transfected plasmid contains a fluorescent marker, positive cells can be screened using a fluorescent microscope or by flow cytometry.
Screening can be done 18h post transfection.