transfection with P/S or without P/S - help advise.. (Dec/19/2005 )
I wanted to know,
1) do cells need to grow in just medium (DMEM) without Strep/Pen before transfection (Hela cells)and during infection ?
2) After how many hours of transfection, its advisable to change the medium and put the medium containing Pen/strep ?
It may depend on the transfection reagent. For example, Lipofectamine from Invitrogen requires no P/S before and during transfection. Transfection medium can be changed after 3 hrs. While Effectene Reagent from Qiagen allows using P/S before and during transfection. If toxicity is a problem, medium can be changed after 6-8 hrs instead of 3 hrs.
I prefer not using P/S at all before and during transfection no matter what transfection reagent I use.
i have lipofectamine from invitrogen and Hela cells to transfect.
If my understanding is correct, i need to plate cells in medium + FCS and without P/S a day before transfection.
and on the day itself, again plate the cells in medium +FCS and without penicillin and streptomycin, do the transfection and change the medium after 6-8 hrs.
please correct if i am wrong.
You are almost right.
But, on the day of transfection, do not plate the cells as you said, simply change medium.
The best thing you can do is to follow whatever on the protocol of Lipofectamine. You may need reduced-serum medium such as Opti-MEM medium during transfection.
On Invitrogen's website there's an optimised protocol for several transfection reagents for Hela cells...