Day 1:
Seed cells -> should reach approximately 20% confluency the next day (e.g. use 150 μ l (from confluent p10 taken up into 10 ml) per well. 2 ml DMEM per well). It is important that the cells are not clumped together. Use trypsin for at least 5 mins and pipet cells up and down against the dish several times to avoid cells clumping.

Day 2:
Transfection #1 per well:
30-60 min prior to transfection replace DMEM with 1 ml fresh DMEM per well.
75 μ l OMEM 2.5 μ l SilentFect (BioRad). Mix gently by pipetting 3x. Sit RT 2-5 mins.
75 μ l OMEM 1.1 μ l siRNA (20 μ M stock => 20 nM final). Mix gently by pippetting 3x.
Combine siRNA and transfection solution. Mix gently by pipetting 3x. No bubbles!
Sit @ RT for 20 mins.
Add dropwise to cells
Incubate O/N

Day 3:
Replenish cells with fresh DMEM (1-2 ml). Be careful with 293 cells. Make sure to preheat and “pre-carbonize” media (omit Pen/Strep!) in a P10 dish in the incubator for 30-60 mins.

Day 4:
Transfection #2 per well:
75 μ l OMEM 5 μ l Lipofectamine 2000. Mix gently by pipetting 3x. Sit RT 5 mins.
75 μ l OMEM 1.1 μ l siRNA (20 μ M stock => 20 nM final). DNA of interest up to 1 μ g.
Combine siRNA and transfection solution. Mix gently by pipetting 3x. No bubbles!
Sit @ RT for 20 mins.
Add dropwise to cells
Incubate for 48 hrs and assay.
Nota bene: If cells look slightly “worn” at the time of the second txn, exchange media after 24 hrs and incubate for another 24 hrs and assay. Be careful when using 293 cells, they come off easily!