3 replies to this topic

[Curtis]

- Is Opti-MEM really necessary? Because I use DMEM and I have 50% efficiency.

- After addition of Lipofectamine to Plasmid, do we need to vortex the mixture vigorously? or just pipetting up and down would be enough?

- Do we need to rotate the plate once in a while after transfection?

- Do we need to remove lipofectamine after 4-6 hours post-transfection and add fresh media?

- Is 16 hours post-transfection enough for cell lysis to detect GFP expression by western blot? what is the recommended time?

Edited by Curtis, 14 June 2010 - 01:04 AM.

[labrat612]

- Be sure to make your DNA: Lipofectamine complex in serum-free media. Opti-Mem might increase your efficiency slightly in certain cell lines. But I've used it in CHO and found that it increased only slightly.

- I personally like to vortex. Nothing too long or too fast.

- Not sure what you mean by "rotate the plate"

- If you are noticing that there is some cytotoxic effects in your cell line, then you may consider replacing the media.

- I would typically wait the full 24 hours before lysis. Check to ensure that you have a decent enough transfection efficiency and then proceed.


~Hope that helps!
Labrat612

[Curtis]

labrat612, on Jun 14 2010, 06:27 AM, said:

- Not sure what you mean by "rotate the plate"

Labrat612

Thanks so much,
I mean rotate or swirl the plate so that the lipo-DNA complex spreads evenly everywhere.

[medchemgirl]

I find that Opti-MEM is necessary. 50% efficiency is pretty low.  And  like the other person said, you have to always use serum free media. and yes, you can incubate up to 6 hrs, because the lipofectamine is toxic to the cells and replace it with SERUM-FREE media.
I do leave them 17 to 18 hours cause then they start dying, but if your cells can stay alive for 24hrs you can try that. The other thing is that u don't want to give the cells the chance to get rid of the new plasmid. Remember, this is transient transfection, and upon cell division the cells will loose the plasmid.

Curtis, on Jun 14 2010, 02:11 AM, said:

- Is Opti-MEM really necessary? Because I use DMEM and I have 50% efficiency.

- After addition of Lipofectamine to Plasmid, do we need to vortex the mixture vigorously? or just pipetting up and down would be enough?

- Do we need to rotate the plate once in a while after transfection?

- Do we need to remove lipofectamine after 4-6 hours post-transfection and add fresh media?

- Is 16 hours post-transfection enough for cell lysis to detect GFP expression by western blot? what is the recommended time?