siRNA transfection - (Aug/05/2007 )

Hello,

I am going to transfect cells with 100nM si-RNA using lipofectamine 2000. How much lipofectamine should I start with. I understand i will have to optimize the lipofectamine amount.

Thanks

here is my protocol for duplexes.
Worked very nice :
J-1 : plate seeding
J0 transfection :
for hela cells 500000 per well in 6well plate
100 000 for Huh7 in 35mm petri dish

final transfection volume 1ml
siRNA concentration : 125nM (stock at 20µM)


Mix 1 : for N dishes
siRNA N*6,25µl
plus reagent : N*3.3µl
Optimem : 90.45µl (up to 100µl)
invert the tube, let homogenize for 15'/RT

Mix 2 :
Lipofectamine N * 12,5 µl
OptiMEM N * 87,5 µl (qsp 100 µl)

Mix mix1 and mix2, invert tube several times, incubate 15'/RT

Trnasfection :
wash cells twice (rinse with pbs or optimem)
put 800µl optimem and add mix of tranfection (mix1+2)

incubate 7°/5%CO2/4hours
Replace medium by medium + SFM

J3 : test by western blot the targeted protein and or northern blot/qRTPCR

Thanks Fred_33. However, isn't the amount of lipofectamine 12.25ul too much?

Also what do you mean by SFM. S= Serum, F?? , M= media
What is plus reagent?
Thanks once again

plus reagent and lipofectamine are both products of Invitrogen. You can use Lipofectamine 2000 single reagent for siRNA. Can't comment on the dosage used for siRNA, but is sounds about right for DNA transfection for that size of plate.

sorry for late reply.
Well this is not too much for DNA transfection.
I used lipofectamine (cheaper than lipo2000) and added plus reagent which is supposed to bind DNA and enhance the liposome+DNA formation. It's a relative cheap reagent that gave me very good results with my exps.

SFM = serum free medium.

Thank you so much.
SFM = serum free medium.
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