Jan 31, 2017

Public workspaceOyster parasite Perkinsus marinus transformation using Amaxa electroporator and non-proprietary electroporation buffer

Nature Methods
DOI
dx.doi.org/10.17504/protocols.io.gv9bw96
  • Imen Lassadi1
  • 1Department of Biochemistry, Cambridge university, UK.
  • Protist Research to Optimize Tools in Genetics (PROT-G)
Imen Lassadi
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DOI: dx.doi.org/10.17504/protocols.io.gv9bw96
External link: https://doi.org/10.1038/s41592-020-0796-x
Protocol Citation: Imen Lassadi 2017. Oyster parasite Perkinsus marinus transformation using Amaxa electroporator and non-proprietary electroporation buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.gv9bw96
Manuscript citation:
Faktorová D, Nisbet RER, Robledo JAF, Casacuberta E, Sudek L, Allen AE, Ares M, Aresté C, Balestreri C, Barbrook AC, Beardslee P, Bender S, Booth DS, Bouget F, Bowler C, Breglia SA, Brownlee C, Burger G, Cerutti H, Cesaroni R, Chiurillo MA, Clemente T, Coles DB, Collier JL, Cooney EC, Coyne K, Docampo R, Dupont CL, Edgcomb V, Einarsson E, Elustondo PA, Federici F, Freire-Beneitez V, Freyria NJ, Fukuda K, García PA, Girguis PR, Gomaa F, Gornik SG, Guo J, Hampl V, Hanawa Y, Haro-Contreras ER, Hehenberger E, Highfield A, Hirakawa Y, Hopes A, Howe CJ, Hu I, Ibañez J, Irwin NAT, Ishii Y, Janowicz NE, Jones AC, Kachale A, Fujimura-Kamada K, Kaur B, Kaye JZ, Kazana E, Keeling PJ, King N, Klobutcher LA, Lander N, Lassadi I, Li Z, Lin S, Lozano J, Luan F, Maruyama S, Matute T, Miceli C, Minagawa J, Moosburner M, Najle SR, Nanjappa D, Nimmo IC, Noble L, Vanclová AMGN, Nowacki M, Nuñez I, Pain A, Piersanti A, Pucciarelli S, Pyrih J, Rest JS, Rius M, Robertson D, Ruaud A, Ruiz-Trillo I, Sigg MA, Silver PA, Slamovits CH, Smith GJ, Sprecher BN, Stern R, Swart EC, Tsaousis AD, Tsypin L, Turkewitz A, Turnšek J, Valach M, Vergé V, Dassow Pv, Haar Tvd, Waller RF, Wang L, Wen X, Wheeler G, Woods A, Zhang H, Mock T, Worden AZ, Lukeš J, Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nature Methods 17(5). doi: 10.1038/s41592-020-0796-x
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: January 04, 2017
Last Modified: March 28, 2018
Protocol Integer ID: 4769
Cell Culture
Cell Culture
Grow Perkinsus marinus at 25 °C in ATCC Media 1886 (see recipes below), until OD600 = 0.4-0.6
Harvest the equivalent of  5-7 107 cells (5 ml at OD600 = 0.5) for the transformation by centrifuging the culture for 10 min at 1000 g at room temperature.
Remove supernatant completely, keep the pellet.
DNA Mix preparation
DNA Mix preparation
In 1.5 ml microcentrifuge tube, add 2.5 µg of purified linearized plasmid + 2.5 µg of circular plasmid, adjust to a final volume of 60 µl using milliQ water.
Add 35 µl of 3R buffer (see recipes below), mix.
Add 10 µl of CaCl2 1.5 M (when you are ready to electroporate)
Electroporation step
Electroporation step
Resuspend the cells pellet with the DNA mix preparation and transfer quickly to the electroporation cuvette (2 mm)
Electroporate ASAP cells using an Amaxa electroporator, program D-023
Resuspend the cells immediately with 500 µl of fresh medium
Transfert the cells to a six well plates previously filled with 2.5 ml of fresh growth medium
Monitor transfection efficiency (by fluorescence microscopy in our case) 24-48 h post transfection.
Buffer and Media recipe
Buffer and Media recipe
3R buffer recipe: 200 mM Na2HOP4 70 mM NaH2PO4 15 mM KCl 150 mM HEPES pH: 7.3 ATCC Media 1886 recipe:
1 Dulbecco's Modified Eagle's Medium Base Sigma D5030 2.10 g
2 Nutrient Mix F-12 Ham Sigma N6760 2.70 g
3 Instant Ocean Sea Water 18.2 g ic-salt/910mL dH2O 400.00 mL
4 L-Glutamine 200 mM 2.50 mL
5 HEPES 1.0 M 12.50 mL
6 NaHCO3 7.5% (w/v) 4.30 mL
7 Carbohydrate Solution 0.5 g Gluc + 0.1g Galc + 0.1 g Treh in 10 mL dH2O 5.00 mL
8 Lipid Concentrate Sigma L5146 0.50 mL
9 Pluronic F68 1 g/100 mL 4.50 mL
10 Fetal Calf (Bovine) Serum   10.00 mL
11 Phenol Red 0.5% (w/v) 0.25 mL
12 Penicillin -Streptomycin Mixture Sigma P0781 5.00 mL
Vacuum filter through 0,2 µm filter pore and keep it on 4 °C for storage (maximum 4 weeks) The preference for P. olseni pH is 7.6 and P. marinus is happy at 7.0 or a little lower (6.8)