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SiRNA Transfection in 60mm plates - (Aug/11/2007 )


How much si-RNA and how much lipofectamine should i add to transfect in 60mm plates. Final volume in plates will be 3ml of media.


considering a 6well plate gave a 3,5cm diameter for a well, i adapted the concentrations for my 10cm plate transfection.
I mean i adapted regarding the amount of cell i plated. In a 10cm plate, i put 4,000,000 cells, so quantities were x8.

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


Don't know if you want to give it a try but...
...I made some experiments with HiPerFect reagent - in my hands it is one of the less toxic reagents for transfection with high efficiency.
The manual provides a protocol for transfection in bigger plates and flasks... .