Transfection help - (Sep/04/2014 )

Hi,

Iam using C3H10T1/2 cells for transfection with X tremegene 9  reagent. The concentration of DNA that I use is 600ng/ well in 24 well plate. The efficiency of transfection is very very low. Do I have to increase the concentration of DNA or do the transfection in 6 well plates. If so how many cells do I have to plate and what is the conc of DNA to be used.

Iam platting 12x104 cells for western in 6 well plate, is this enough or do I have to plate more. Please suggest

Thanks,

Jaya

Hi Jaya,

Roche give some pretty extensive instructions for this reagent which you can get here.

The correct ratio and amount of DNA and reagent varies between cell type.

It's not clear to me what cell density you have been using but the cells need to be 70-90% confluent at the time of transfection. Plate the cells the afternoon/evening before the transfection (for DF-1 I use 1.2x10E5 per well of a 24-well plate).

It might be worth seeing if you can improve efficiency using a brief period of shaking as described here but I haven't yet gotten around to trying that myself.

Thank you. Let me try with the shaking method.

Let me know how it works out! I'm planning on testing this within the next few weeks and will also report my findings to the forum.

Sure I wil.