salam alikum

Highest transfection efficiencies are achieved when the complexes are added to cells in their complete media (serum-containing). However, Some transfection protocols require serum-free conditions for optimal performance, since serum can interfere with many commercially available transfection reagents. This should be checked for your protocol.

important points for the best results in RNA transfection :

1- Cell density at the time of transfection

2-The ratio of RNA to transfection reagent

3- Determine the best incubation time post-transfection for each Jurkat cell subtype. The optimal incubation time is generally 24–72 hours, but will vary depending on the goal of the experiment, the nature of the plasmid, and the half-life of the expressed protein

4- Antibiotics may inhibit transfection complex formation and therefore should be excluded from the complex formation step. Transfection complexes can

be added directly to cells grown in complete culture medium containing serum and low levels of antibiotics (0.1–1X final concentration of penicillin/streptomycin mixture).

5-For the best results and the most efficient transfection, an optimal ratio of DNA to transfection reagent must be determined for each cell line–plasmid combination. Therefore, it is vital that optimization trials are carried out for each new cell line–plasmid combination used.

Please visit the following link it contains useful information

Good Luck

http://www.qiagen.com/sa/resources/molecular-biology-methods/transfection/#Typical number of adherent cells to seed

Thanks so much for the quick response. All the points are very helpful. Yes. I visited the Qiagen website too. Very useful.

what is the mode of transfection and incubation time post transfection in the media. RPMI 1640 can be used but it is to be supplemented with 10%FBS.

Dear Laxmi,

Thanks for your response. It is passive transfection with a reagent up to 2 days. I will most likely do the transfection in antibiotic free medium and after 4 h suppl with regular medium. Yes. I will use medium with serum for the transfection process also.

Antibiotics dont hamper transfection process. Yes serum free media for 4 h will

be better.

ok. that's good to know .Thanks.

The use of serum-free medium during the first few hours of incubation versus complete medium  will also be highly dependent on your transfection reagent or method. Some reagent perform better in presence of serum whereas other not. Moreover, this will also be dependent of your cells are you testing primary T cells or cell lines, in the latest case I can suggest DreamFect Gold or Lullaby for siRNA transfection. if you need sample, let me know [email protected]

Good luck, Olivier

LGM3 medium is used for transfection of human T cells by nucleofection, since you are using some passive method, it may be of help to you. You can purchase it from Lonza company where you have to supplement with glutamine.

Sure. Thanks for the feedback. If the current reagent does not work maybe i will try some of the suggested ones here in this discussion thread.