Invitrogen’s Lipofectamine Reagent Transfection Protocol
Day1: Seed cells in growth medium (with the usual amount of serum)
6-well
60-mm
10-cm
3T3/K41/K42/KB188
2x104
2x105
5x105
HEK293
9x104
medium
2ml
5ml
10ml
9x105
Day2: cells will be 50-80% confluent at the time of transfection.
1. Dilute DNA in Opti-MEM® I Reduced Serum Medium. Mix gently.
2. Mix Lipofectamine™ gently before use, then dilute it in Opti-MEM® I Medium (or other medium)
without serum. Mix gently.
3. Combine the diluted DNA with diluted Lipofectamine™. Mix gently and incubate for 30 minutes at
room temperature (solution may appear cloudy).
4. Remove the growth medium from cells and replace with Opti-MEM® I Medium.
5. For each transfection, add Opti-MEM® I Medium to the tube containing the complexes. Mix gently.
6. Add diluted complexes on the cells. Mix gently by rocking the plate.
7. Incubate cells at 37°C in a CO2 incubator for 5 hours.
8. After 5h of transfection,Remove the transfection mixture and replace it with fresh, complete medium (with
normal amount of serum).
9.For transient transfection: Test for transgene activity 24-72 hours posttransfection as appropriate for
your cell type and expression vector.
10.For stable transfection: Passage cells at a 1:10 dilution into selective medium 72 hours posttransfection.
Culture
vessel
DNA(µg) in
media vol. (µl)
24-well
0.2-0.4 µg in 25 µl
35-mm
1-2 µg in 100µl
60-mm
3-6 µg in 300µl
10-cm
8-16 µg in 800µl
Lipofecta
mine™
(µl) in
media
vol.( µl)
0.5-5 µl
in 25µl
2-25 µl in
100µl
6-75 µl in
300µl
6-200µl
in 800µl
Relative surf.
area vs.24well
1
Replace on
cells new
medium
vol.
0.6ml
Add to
complexes
medium
vol.
350µl
5
1.2ml
600µl
10
2.6ml
1.8ml
30
5.6ml
2.8ml