Problem with gfp express in S2 - (Jul/19/2007 )

Hi, hope someone can give me some advice...
I make a gfp plasmid and i transfected it into my drosophila s2 cells but i see no fluorescence. i wanted to do some RNAi work with this cells but i can't now...
I used fugene in a ratio of 8microL fugene with 1microG of plasmid..
I don't know if its my plasmid that didn't work or my transfectio, how can i check which one don't work?
How long must i wait after transfection to be able to see the green fluorescence?

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I'm no expert, have only done transfection once before (and not very successfully I should add!!!) But you could:

Try transfecting an easily transfectable cell line (maybe COS7??) with your plasmid to see if you get fluorescence??

Also, try titrating a few different amounts of plasmid, I have heard getting the right amount can sometimes be very important.

Do you have a selection marker also??

I would wait 24 hours before checking for fluorescence.

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Which promoter are you using (in front of your gfp)? How do you check fluorescence? Microscope? FACS? Selection marker on your plasmid as well?

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If your prepared DNA using a maxi kit, then usually its fine. Also check it by running on a gel.

You have to optimize the transfection and this might take a couple of trials. But if you do get a successful transfection, you could see GFP fluorescence in 24 hours.

One important thing is to know your promoter works in these cells. If they dont work then you might have a tough time to evaluate transfections.

Good Luck !!!

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thanks alot

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Hi guys thanks for all the rely,
i will try different amount of plasmid for transfection.(finger cross)
The promoter shouldn't be a probem as i used an old luciferase plasmid that work,
swap the luciferase with GFP.
I did a maxi and sequenced my plasmid to confirm

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I transfected S2 using commercial reagents but got bad efficiency. Then, I used home made calcium phosphate, and it worked fine.

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Hi
We have successfully transfected a GFP plasmid into S2 drosophila cells using Lipofectamine plus from Invitrogen. I used to use Calcium phosphate to transfect these cells, but lipofectamine is both easier and more efficient.
Send me a PM if you'd like some of our plasmid.

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