Jun 05, 2024

Public workspaceTransfection of mammalian cell lines with plasmids and siRNAs

DOI
https://dx.doi.org/10.17504/protocols.io.261ge55byg47/v1
  • Agnes Roczniak-Ferguson1,2,
  • Shawn M. Ferguson1,2
  • 1Departments of Neuroscience and of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Berrak Ugur
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DOI: https://dx.doi.org/10.17504/protocols.io.261ge55byg47/v1
Protocol Citation: Agnes Roczniak-Ferguson, Shawn M. Ferguson 2024. Transfection of mammalian cell lines with plasmids and siRNAs. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge55byg47/v1
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 24, 2024
Last Modified: June 05, 2024
Protocol Integer ID: 101278
Keywords: ASAPCRN, transfection of mammalian cell line, mammalian cell line, plasmid, sirnas this protocol detail, transfection, sirna, line
Funders Acknowledgements:
ASAP
Grant ID: 000580
Abstract
This protocol details the transfection of mammalian cell lines with plasmids and siRNAs.
Troubleshooting
Lipofectamine 2000 (Invitrogen) or Fugene HD (Promega) or Fugene 6 (Promega) transfection reagents
20m
On the day before transfection, plate 100,000 HeLa cells per well in a 6 well dish. For other cell lines, the number of cells will need to be optimized to achieve 50-75% confluency on the day of transfection.
Warm Optimem (Gibco) and transfection reagent to TemperatureRoom temperature
Temperature
Add Amount200 µL Optimem + Amount6 µL transfection reagent + Amount2 µg plasmid DNA to a round bottom polystyrene tube and mix gently.
Pipetting
PCR
Incubate Duration00:20:00 at TemperatureRoom temperature .

20m
Incubation
Add dropwise to cells and mix gently. Return cells to incubator. Duration of transfection must be optimized for each plasmid and downstream application.
Pipetting
Mix
RNAiMax transfection reagent
20m
Warm Optimem (Gibco) and RNAiMax (Invitrogen) transfection reagent to TemperatureRoom temperature .

Temperature
Combine Amount200 µL OPTiMEM, Amount5 µL RNAiMAX transfection reagent and Amount5 µL of Amount20 µL siRNA and mix gently.

Mix
Incubate Duration00:20:00 at TemperatureRoom temperature .

20m
Incubation
Add transfection mix to Amount1.8 mL media containing 100,000 HeLa cells per well in 6-well dish and return to cell culture incubator for 24-72 hours (duration needs to be optimized for each target gene).

Incubation
Mix