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Early transfection


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#1 robradford

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Posted 13 July 2010 - 07:23 AM

Hi everyone,

I'm trying to transfect my cell line with GFP-alpha tubulin, and so far in our lab we haven't been able to transfect our cell line at all (it's a proximal tubular epithelial cell line, which is super adherent, and grows in very very tight colonies).
So we have tried a few different protocols but have had no luck. I heard someone mention in passing though that I should transfect as I seed my cells as this gets around the problem posed by the colony formation.
Does anybody have any opinions on this? Can anybody see a reason that I'm not seeing as to why this wouldn't work (I'm very new to all of this transfection malarchy!). Is there anything to take into condsideration specifically arising from transfecting so early?
Any help is very much appreciated.....as I said, I'm pretty green when it comes to this sort of work :)!

Cheers,

Rob

#2 bob1

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Posted 13 July 2010 - 06:02 PM

"reverse transfection" (transfecting as you are seeding) works quite well for most cell lines that I have done it on, but then I only really use pretty common cell lines for most of my stuff.

It may be a problem if you are trying to transfect something that will interfere with cytoskeletal or cell cycle processes.

#3 robradford

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Posted 14 July 2010 - 12:15 AM

"reverse transfection" (transfecting as you are seeding) works quite well for most cell lines that I have done it on, but then I only really use pretty common cell lines for most of my stuff.

It may be a problem if you are trying to transfect something that will interfere with cytoskeletal or cell cycle processes.


Thanks bob1, a friend of mine has used the GFP tubulin in another cell line and it didn't seem to interfere with the cytoskeleton, but I suppose he didn't transfect in this way (thanks for giving me the proper name for it by the way!). Sure I'll try it and I'll be vigilant looking for any alterations to the cytoskeleton compared to untransfected cells.

Thanks again for your help bob1,

Rob

#4 bob1

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Posted 15 July 2010 - 06:57 PM

Ah, I should have read your post a bit more carefully and seen the tubulin. Reverse transfection should be fine for this - I was thinking more if you were transfecting something like pRB or p53 that affects the cell fate - then reverse transfection can be a problem.

#5 laurequillo

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Posted 15 July 2010 - 09:11 PM

Ah, I should have read your post a bit more carefully and seen the tubulin. Reverse transfection should be fine for this - I was thinking more if you were transfecting something like pRB or p53 that affects the cell fate - then reverse transfection can be a problem.


Hi there! Ey Bob, could yo tell me how you do the reverse transfection? Do you some how fix the dna in the plates, or you just do the transfection at the same time that you seed the cells? And how good is the efficiency?
I never used it, and I would like to give it a try!
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#6 bob1

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Posted 16 July 2010 - 04:42 PM

If you are using lipofectamine or one of the many variants, then the protocol is on the product sheet. Basically it is just mixing up the transfection mix and adding it the wells/dish/flask and then adding the cells and mixing.

#7 robradford

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Posted 18 July 2010 - 11:29 PM

Ah, I should have read your post a bit more carefully and seen the tubulin. Reverse transfection should be fine for this - I was thinking more if you were transfecting something like pRB or p53 that affects the cell fate - then reverse transfection can be a problem.


I'll be starting this week, so thanks again for the advice bob1!

Rob




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