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