TITLE: Electroporation of the protist Cafeteria roenbergensis cells using the Neon Transfection System
AUTHORS: Sarah Duponchel, Monica Berjon-Otero, Matthias Fischer

[DESCRIPTION]
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[STEPS]
?. [Cell preparation]
Before the experiment (it can be the day before without affecting too much the cell viability)
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?. [Cell preparation]
Centrifuge the Cafeteria cells at 4,500 g, 5 min, room temperature
?. [Cell preparation]
Wash twice the cell pellet with 1X PBS=> use small volume of PBS to concentrate the cells
?. [Electroporation buffer preparation]
Prepare the different reagents (CaCl2 and 2X HEBS) according to the followed protocol from Pia Elustondo
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Aliquot and autoclave the 2X HEBS buffer to avoid contamination
?. [Electroporation buffer preparation]
Mix the CaCl2 (final concentration 125 mM) with the material to electroporate- for DNA electroporation complete to the final volume with water- for protein electroporation complete to the final volume with 1X PBS or the buffer the most suitable for the proteinFor one electroporation, final volume of the CaCl2 mixture10-μl tips: 3.5 μl100-μl tips: 48.5 μl
?. [Electroporation buffer preparation]
Add the CaCl2 mixture onto the 2X HEBSFor one electroporation, 2X HEBS volume10-μl tips:  5 μl100-μl tips: 50 μlOption 1: volumes are big enough to vortex constantly  the HEBS while the CaCl2 mixture is added drop by dropOption2: 1/10th of the CaCl2 mixture is added at a time with a 3-secondes vortexing-step between each addition
?. [Cell preparation]
Count the cells: stain 10 µL of Cafeteria culture with 1 µL of Lugol’s acid iodine solution and load them in a haemocytometer (Neubauer Chamber)Maximum volume of the desirable number of cells needed for the electroporation = 1.5 μl
?. [Electroporation buffer preparation]
Add the 1.5 μl volume of the appropriate number of C. roenbergensis cells to the CaCl2/HEBS mixture
?. [Electroporation]
Electroporate the cells at 1600V, for 20 ms, 1 pulse using either the 10-μl or 100-μl tips
?. [Cell recovery]
Place the electroporated cells into 10 time more f/2 media than the electroporated volume for 30 min
?. [Cell recovery]
Cells can either be fixed or maintained in culture by adding 5 time fresh f/2 media complement with 0.02% yeast extract and 1/500th of E.coli culture
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