I made transfection of some cell lines using siRNA and cationic polymer, I am sure the polyplexes were formed (NP ratio of polyplexes were determined by the agarose gel) but the transfection efficiency is very low (5-25%).
I wonder which sequence is better for the transfection:
1- Removing the media from the plate wells, washing the cells by PBS, adding serum free media, then adding the polyplexes solution.
Or 2- I should mix the serum free media with polyplexes solution first then add them to the plate wells after washing with PBS.
Does washing the cells by PBS before the transfection affect the transfection?
Hi Mai,
- Both adding orders would work; however, I prefer to go with the first one.
- Check the instructions of your transfection reagents. Sometimes, it is recommended to wash the cells with 1XPBS before the transformation. If that the case, make sure the 1XPBS is sterilized and warmed up to 37C before adding. On the other hand, you should wash your cells with 1XPBS to get red of the serum.
- Sometimes, leaving the cells in the new medium (in your case, serum free) for few hours (1-6h) before transfection could increase the transfection efficiency.
Good luck
Hi Mai,
Probably I agree that both cases should work. But in case they didn't gave you satisfactory transfection you may troubleshoot as follows:
1. Revive a lower passage cell line and try transfection.
2. Timing is impotant : if you remove Sera from cells, you should wash cells within 6 hours of transfection or in sera-case, you can transfect your cells for 24h maximally.
3. Repeat transfection with lower cell seeding no.
4. Have you ever tried CaCl2 mediated transfection?
Thank you Pratibha and Ahmed for your replies.
I tried the transfection with lipofectamine 2000 and it worked good (~60% efficiency) but when I try to transfect using pegylated poly-L-lysine polymer, I get 0-10% transfection efficiency.
After 4h of transfection in serum free media, I add media with 20% FBS. Do you think it's necessary to wash the cells and add media with 10% FBS?
Do you mean CaCl2 or Ca3(PO4)2 transfection?
I would agree, that Lipofection would be potentially more effective for transfection of cells with small nucleotide sequences. Maybe, the cell type you use is not easy to transfects, such as most primary cells. In this context, electroporation would be an alternative offering an increased transfection efficacy, but also an increased death rate, due to the method itself.
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