Mar 08, 2017
Version 1
Transfection of Alexa488-labelled DNA into Oxyrrhis marina with polyethylenimine (PEI) V.1
- Protist Research to Optimize Tools in Genetics (PROT-G)
- Slamovits Lab
External link: https://doi.org/10.1038/s41592-020-0796-x
Protocol Citation: Pia Elustondo: Transfection of Alexa488-labelled DNA into Oxyrrhis marina with polyethylenimine (PEI). protocols.io https://dx.doi.org/10.17504/protocols.io.ha8b2hw
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: March 08, 2017
Last Modified: November 19, 2017
Protocol Integer ID: 5184
Keywords: dna into oxyrrhis marina, labelled dna, alexa488, dna, polyethylenimine, transfection
Attachments
PEI Alexa488DNA.pdf
386KB
Troubleshooting
1. Reagents
Polyethylenimine (1 mg/ml ) (Polyscience Cat # 23966):
Heat 50 ml of water to 80 C
While stirring, add 50 mg PEI stir and dissolve with heat on. Once dissolved let it cool to RT and pH to 7.4 with HCl
Filter in hood, aliquot and store at - 80 C
Avoid freeze thaw cycles, Discard unused once thawed.
2. Prepare transfection mix
Per 500-1000 μl of culture to transfect:
F/2 media 200 μl
DNA 1-5 μg
PEI 2-3μl per μg of DNA
Incubate at RT for 30 min
3. Add the mixture to the cultures
Add PEI/DNA mix to the culture.
The next day the culture can be washed with fresh media with adequate supplements.