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Basic questions about Transfection


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3 replies to this topic

#1 Curtis

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Posted 14 June 2010 - 12:11 AM

- 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.


#2 labrat612

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Posted 14 June 2010 - 06:27 AM

- 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

#3 Curtis

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Posted 14 June 2010 - 06:58 AM

- 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.

#4 medchemgirl

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Posted 14 June 2010 - 12:52 PM

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.

- 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?






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