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DNA Techniques Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


Hello to everybody!

Hello to everybody! - DNA Techniques

Hello to everybody! - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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  #1  
Old 08-04-2006, 08:43 PM
Pipette Filler
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Default Hello to everybody!



hi to all!

This is my first email, I decided to join this website to find someone who can help me!!

I do the transient transfection of OKP cells (two genes) using lipofectamine (5ul per well..in 6well plates) then I grow again the cells for nearly 15h and finally I place medium without serum for 24 h. The problem is that 48 h after transfection (when the cells should be ready for the experiment) my cells die!!
What's the problem?
The toxicity of lipofectamine? The kind of quiescent serum (Low glc DMEM plus antibiotics) or what?
Please help me.

Thanks a lot

mimmy
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  #2  
Old 08-04-2006, 09:58 PM
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Default Re: Hello to everybody!

Dear Mimmy,

Hello and welcome to the forums!

From what I can tell by reading your post, you are leaving the cells very long in serum-free medium. These OKP cells what type are they?

I would try transfection with Fugene-6 which does not need changing of media or serum-free media. your cells will definitely do better with the media in place at least while you transfect. Lipofectamine is toxic although it shouldn't be that toxic, however with 24 hours without serum that is not so good.

Try to optimize your transfection conditions, use Fugene-6 or other transfection reagents allowing serum media keeping your cells healthy!

Good luck and make sure you tell us how you are doing soon!
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  #3  
Old 08-14-2006, 09:56 PM
Pipette Filler
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Default Re: Hello to everybody!

Thanks a lot moleculardude,

for now I am trying to reduce the hours of incubation after transfection and it looks like better!
I have never tried Fugene-6, if could be a good alternative!

Thank you
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