Feb 16, 2025
Transfection of HEK cells with Lipofectamine 3000
- 1Caltech
Protocol Citation: Chelsie Steele, Yujie Fan 2025. Transfection of HEK cells with Lipofectamine 3000 . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr97wzvmk/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: January 15, 2025
Last Modified: February 16, 2025
Protocol Integer ID: 118482
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
how to transfect HEK cells with lipofectamine 3000
Materials
Lipofectamine 3000 - Lipofectamine 3000
Opti-MEM reduced serum medium - Opti-MEM reduced serum medium
Cell preparation - Day 0
Cell preparation - Day 0
Plate HEK cells in 24 well plate: 100K per well
Day 1
Day 1
Check cells --> less than ~60-70% confluent wait another day, if 60-70% confluent can begin lipofectamine transfection
Lipofectamine Transfection
Lipofectamine Transfection
15m
15m
Be sure to measure your DNA concentration before beginning the transfection - as you want a concentration of 0.5-5 ug/ul. Dilute accordingly
Calculate how many wells should be transfected. The below graph is the amount of materials needed to transfect 1 well in a 24 well plate. Multiply the materials accordingly depending on how many wells needed.
Lipofectamine Transfection components
Prepare 2 tubes per plasmid. One tube will have Opti-MEM reduced serum medium medium with the lipofectamine 3000 reagent and the other will have Opti-MEM reduced serum medium with the plasmid and P3000 reagent (see above graph)
Add the solution from tube 2 into tube 1 and mix well
Incubate the mixture for 10-15 minutes at RT
15m
Add 50 ul of the mixture from step 7 to each well. Gently swirl plates to ensure homogenous distribution of complex to the entire well