Transfection of 293 cells for protein expression - optimum conditions for transfection!!! (Sep/08/2006 )

Hi guys,

I have different constructs that I generated. some of the should encode secreted protein while others don't. I want to confirm protein expression in vitro using HEK 293 A cells, so I can go further into my in vivo studies.

my plasmids are pcDNA3.1 and I'm using Fugene 6 for traansfection.

1. I prepared my plamids using maxi prep from Qiagen (Do I nedd endotoxin free reagents???)
2. I grew my cells in antibiotic free media (50 to70% confluent) in 6 well plate.
3. I linearized my plasmmid (I used only one of them just to optomize my conditions at the
beginning) then I will go with the others.
4. I linearized my control plasmid pcDNA/CAT.
5. I transfected my cells with plamisd complexes using 7 ug of the linearized plamisds (my plasmid
and CAT) and as a control I used supercoild plasmids to compare. I didn't change the media
and I used serum free media with fugene 6.
6. I collected the media as well as the cell lysate to check protein expression using western blot
at three time points 24, 48 and 72 hours.

--can anyone tell me if I have to do something diffrent than what I did.
--Should I change the media before transfection? and wash the cells with PBS??
--should I use endotoxin free media?
--I used water to elute my plasmids using maxi prep, is that ok??
--my protein should be secreted in this case, do I have to do anything else other than what I did?


another question when I linearized my plasmid I used PCR purification kit from Qiagen but I lost most of my plasmid ( Ilost around 30 ug) !!! Should I heat inactivate the enzyme instead??


Thanks in advance,

No need to change media before transfection or even wash them. I usually dissolve my maxi DNA in TE.

I think the right control will b linearised plasmid.

I think for Fugene, u need only 2 ug of DNA per well in a 6 well plate. So please verify ?

I dont linearise my plasmid before transfection and still get high transfection efficiency.

u could heat inactivate the digested plasmid or even gel purify the DNA or just precipitate the DNA.

Agree with scolix's suggestions.

"--Should I change the media before transfection? and wash the cells with PBS??
--should I use endotoxin free media?
--I used water to elute my plasmids using maxi prep, is that ok??
--my protein should be secreted in this case, do I have to do anything else other than what I did?"

- You can grow cells with antibiotics, no changes needed before transfection, no wash needed, just remove the old medium and add complexes to cells.
-if only for in vitro screen purpose, no need to use endo-free kits. you may need it for in vivo studies.
-water elution is fine, as long as you freeze it after purification. TE is most peoples' choice.
- collect supernatant at time point indicated and collect cells at the end as well to see if proteins remain in cells, just in case.

Thank you guys very much,

I added my plamisd complex directly to the media I didn't remove the media before transfection !!

should I remove the media as genehunter mentioned or it is ok to add it directly to the old media ??

Thanks again

I dont change media for fugene transfection. I add the DNA complex solution to the cells.

Hello,

please consider if your cells have the LPS receptors (TLR-4 & CD14) or not. You should use endotoxin-free materials if your cells could react on LPS contamination.

If so you should use pyrogen-free plastic material and also pyrogen-free water.

Yours Sincerely,
Stephanie

We provide more information regarding endotoxin on our webpage: www.endotrap.de