MAMMALIAN CELLS TRANSFECTION PROTOCOL(Javier Calvo)

Follow invitrogen lipofectamine 2000 protocol

http://tools.invitrogen.com/content/sfs/productnotes/f_lipofectamine%202000b-040923-
rd-mkt-tl-hl050602.pdf

Tips:
The day before plate cells at 4-5x106/p100 to get the following day 80-90% confluency.
Recomendations: Highest transfection efficiency in cells at  90% confluency.
Use a range of several ul of lipofectamine and ug DNA to increase viability after
transfection and efficiency.

For a p100 (according to Invitrogene):

1. Mix OPTIMEM with DNA in an eppendorf (5-10 g DNA-0,8 ml OPTIMEM)

2. Mix OPTIMEM and lipofectamine in another eppendorf (32 l lipofectamine in 0,8
ml OPTIMEM)
3. Incubate 5 minutes at RT.
4. Mix both eppendorfs and incubate 20minutes.

During the 20minutes:

Remove medium from cells.

Wash twice with PBS.
Add OPTIMEM (6,4 ml).

5. After 20minutes add mix coming from OPTIMEM+DNA and

OPTIMEM+LIPOFECTAMINE to cells with OPTIMEM (1,6 ml).

Incubate 4hours, never more than 6hours.

Remove medium.
Add fresh growing medium.

The amount of DNA/OPTIMEM/Lipofectamine can be adjusted to lower the cost:

For example:
Use 0,36 ml OPTIMEM + DNA
0,36 ml OPTIMEM +14 l lipofectamine
Use 2,5 ml of OPTIMEM in the p100 (so after adding the DNA the cells are covered by
only 3,2 ml of media, which is the minimum amount)