Apply tranfection reagents DIRECTLY onto the cells. - After removing the medium. (Jan/22/2007 )
i think i became confused after the talk with my PI. I was tought and read that I add transfection reagents (ca phos precipitates, PEI precipitates) into the medium and shake the cells. when i told this to me PI he was he said "NOWAY! that reduces the efficiency, you should remove the medium and put transfection reagents directly onto the cells. and after some incubation time add some medium. when i told him that i didnt read this anywere, he said "because it is a secret. noone tells you to do that".
anyway yesterday i tried to do so and cells (neurons) seem ok, still didnt check the transfection effeciency though. any comments?
Did you mean adding complexes made in diluted DNA and transfection reagent directly to cells, right? I have done both ways and found the difference is minor if you mix it with cells well. Sometimes when you have to work with multiple wells, you'd rather to add some medium to cells before you lay the mixture onto cells, or cells will be dried out. Let us know your experience.
We used to add the DNA and transfection mixture to the cells after removing the media. And we would add more media to it or replace it later.
If u plan to add the transfection mixture directly to the media , make sure the final volume is within limits and not diluting the mixture more than necessary.
thank you for your replies, guys. just to update i am trying both ways and well maybe adding reagents directly onto the cells and shaking them at 37 for some time makes some difference... actually the only time i got positive neuron was when i incubated them for 30 minutes. i am also trying to see if centrifugation has some effect. doesnt seem so though.
adding transfection mixture directly on cells is done in my op, for high concentrations of Lipid/DNA complexes on cells.
Incubation time should be decreased (you sais it was 30') in comparison to greater solutions (i let my siRNAs for 5hours...)
it's feasible with small culture vessels (you can't do that in 15cm well plate, or you are super rich lab for puchasing enough lipids )
so general rules are with medium as it's the more used trnasfection way
I said again these are from my thoughs, and not scientifically tested.