this is my first time doing siRNA transfection
I just would like to know if I am seeding a 6 well plate with cells do I use complete media or optiMEM?
Also upon treatment of the cells do I add 250 microliter of the formulated complex of siRNA/lipofectamine to the well without removing the complete media or I remove the media first and then add only 250 microliter of the formulated complex of siRNA/lipofectamine and incubate?
My last question what is the final volume to be present in a 6 well plate is it 2 ml or 5 ml?
thank you so much for the help
Seeding of cells is done in Complete growth media (2ml/Well) , usually one day before transfection. OptiMEM is used only for dilution of siRNA and Lipofectamine RNAiMax reagent.
You can add the 250µl RNAi complex directly to the cells without change of media.
I would recommend incubate the RNAi treated cells for 72h to get the highest knockdown effeciency.
Good luck!
Seed your cells with complete media, but make your transfection complexes with Opti-MEM. You should remove ~50% of the media from the cells the next day before adding your transfection complexes.
The reason you don't use complete media when making the complexes is that the FBS inhibits the formation of the transfection complexes.
Best of luck :)
one last thing is it okay to add antibiotic in the media or preferably not as I read that antibiotic causes cell death?
preferably not, as antibiotics may affect the process of endocytosis. since lipoplexes are internalized via endocytosis this may affect uptake and transfection efficiency.
If I am going to incubate the siRNA treated cells for 48 hours do I need to change the media?
if I need to change the media do I change it with complete media only or with media containing siRNA/lipofectamine complex?
thanks in advance
yes, you need to change the medium. typically this is done after 4 hours of transfection, but you may decide to keep your siRNA complexes for longer time. In that case you add an equal volume of complete medium without removing the transfection medium. After 24 hours you then change the medium completely.
There are many things to be considered, including cell type and ease of transfectability, and half life of your target protein. If half life is short you may want to add siRNA complexes twice.
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