Transfection in 6-well plate
Seed cells in 6-well plate, seed ~1e6/well and culture for overnight.
Prepare transfection mixture as below:Dilute 10ul Lipofectamine 2000 in 125ul Opti-MEM, mix well and incubate at RT for 5min.
Dilute 2.5ug plasmid in 125ul Opti-MEM, mix well and incubate at RT for 5min.
Add diluted Lipofectamine to diluted plasmid, mix well and incubate at RT for 10min.
Change 1.5ml/well serum free medium (or Opti-MEM)
Add transfection mixture onto cells.
4-5h after transfection, change 2ml/well complete medium.
Notes
The amount of transfected plasmid can be varied from 2ug to 4ug.
The amount of Lipofectamine 2000 reagent can be varied from 5ul to 12ul.
The amount of Opti-MEM can be veried from 125ul to 250ul. (final transfection mixture volume 250ul to 500ul).
The transfection mixture must be made in Opti-MEM, but the mixture can be added directly to cells in culture medium with serum and antibiotic.