Transfecting 293 cells using oligofectamine - (Oct/10/2005 )
I am trying to transfect some siRNA oligos into 293 cells using Oligofectamine.
I am using an invitrogen protocol that doesn't specify whether the well should contain media before the complexes (siRNA : Oligofectamine) are added to them.
Does anyone have a standard protocol that I can use?
I have transfected siRNA into 293 cells using lipofectamine 2000 from Invitrogen and found that 293 is sensitive to lipofectamine. This is how I do the transfection. In a 6-well pate, I plate 293 cells at a density of 70-80% in 2 ml medium without antibiotics. The following day do the transfection by adding 500 ul lipo/siRNA complex directly into each well (make sure adding the complex drop by drop to differnt area of the well), and 3 hours later, I replace the transfection medium with fresh medium w/o antibiotics. For other cells, I don't usually change medium after transfection.
Hope that helps.
Alternatively, you may just use calcium phosphate for 293 cells (so cheap...)
If you do so, transfect your siRNA twice (24 hours after 1st transfection)