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Transfection before plating?


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#1 DocClever

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Posted 11 February 2010 - 09:16 AM

Hi!

Does anybody know a protocol how to transfect dissociated neurons before plating? I don't mean electroporation but some other technique like e.g. lipofectamine.

Somehow in the depths of my brain i remember having seen a protocol how to do this but if it really exists i can not find it any more.
The point is that i'd like to transfect neurons plated on glial feeder layers but the Ca-phosphate protocol i use gives me a tremendous background of nicely transfected glia cells. So if i could find a way to transfect the cells before plating, this would be great.

Any comments are appreciated!

Best,
Tom

#2 bob1

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Posted 11 February 2010 - 03:25 PM

It's called a reverse transfection - lipofectamine2000 has the protocol on the product sheet. Basically, mix your DNA and lipofectamine dilutions, incubate for 20 min, place in wells, add cells. In your case, you could add to the cells in a tube for a few hours, then seed out. I am not sure how the cells will survive being in a suspension for an extended period though.

Edited by bob1, 11 February 2010 - 03:27 PM.


#3 DocClever

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Posted 12 February 2010 - 02:18 AM

Thanks!
Basically this is my plan. I made the experience that i get nice expression already after 25 min of incubation with lipofectamin/DNA. So i hope i'll have enough cells that survive this procedure and get transfected.
I will post my results here.

Regards,
Tom




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