I'm trying to ascertain the volume of the transfection mixture in which the cells are incubated for transfection to occur. I consulted a few documents/protocols (links below) and what I understand is that 250ul of final mixture is to be added to each well (which may be replaced after 4-6 hours). However, if that's all the media that's to be added, 250ul looks like insufficient and I am afraid the monolayer will dry up and cells detach if I use this much less amount of medium. Are my concerns genuine? How do you go about the process in your lab? If I add additional 750ul of Opti-MEM per well in one of my experiments, I think the mixture will be diluted and thus won't work. Any suggestions?
1. http://personal.psu.edu/drc9/data/Product_Inserts/lipofectamine2000_man.pdf
2. http://people.ds.cam.ac.uk/yhbl2/files/protocols/lipofectamine2000.pdf
3. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Lipofectamine_2000_Reag_protocol.pdf
Grow your cells in 6 well plate in 2ml of your media of choice until they reach >90% confluency. Change the medium for fresh one just before the transfection (2ml/well).
Protocol for one well:
- Tube #1: Dilute 4ug of your DNA in 250ul of Optimem.
- In the meantime mix Lipofectamine with Optimem - 10ul to 250ul in Tube #2 and let it incubate for 5min RT.
- After 5min incubation gently mix both tubes (DNA and Lipofectamine) and incubate for 20min RT.
- After 20min incubation add whole 500ul directly to the wells with cells in 2ml medium.
Always add the final transfection mix to the cells in the wells with medium.
Link #1 that you provided gives you a nice table with scaling up and down.
Dear Wassem,
In here we seed the cell in 60-70% confluence about 1000000 cell/well in 6-well plate and in second day we transfected the cell by adding 300ul of the transfection mixture and we used 10-15ul of Lipofectamine-2000 and 2.5ug of DNA and we culture them for 36-48 hrs more then harvest the cells. this protocol work very well with 293T cells. in the second day of transfection we don't change the media as 293T is very easy to be attached.
Regards,
Ahmed
The low amount of volume is to allow for maximum contact between your DNA-lipid complexes and the cells. If you have a good incubator setup (that minimizes evaporation), 250ul will be more than enough to cover the entire monolayer of cells. I've done it with 200ul - just swirl it around gently. However, if you don't incubate your cells in the optimal conditions, there will be evaporation and your cells will dry up. Of course there are other factors such as quality/position of the plate in the incubator (e.g. if the plate is not flat then obviously 250ul will not be sufficient to cover the whole well). Hope that helps!
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