1
Seed
400 000 cells per well of a 6-well plate (40 000 cells/ cm2)
2
Next
day: (cells should be 80% confluent): Prepare transfection mix:
Per well (per 1 ml of transfection mix = for 10 chambers of an 8-chamber
LabTek):
2.1
Dilute
the DNA in 100 µl serumfree medium, add Plus-reagent and mix (do not vortex).
Incubate for 15 min at room temperature.
(The total amount of DNA should be the same for all samples; if necessary add
an unrelated control plasmid. If you want to control the transfection
efficiency, you can add a constant amount of a GFP-vector, e.g. EGFP-C1 from
Clontech).
2.2
Dilute
the Lipofectamine in 100 µl serumfree medium.
2.3
After
the incubation of the DNA solution with the Plus-reagent, add the Lipofectamine/medium
solution (100 µl), mix carefully and incubate for 15 min at room temperature.
2.4
Add
800 µl serumfree medium (gives a total of 1 ml) and add the transfection mix to
cells, which have been washed with serumfree medium shortly before.
2.5
Incubate
the cells for the appropriate time at 37°C in the incubator (incubation time
depends on the cell type).
2.6
After
the incubation, remove the transfection mix and add the normal serum-containing
medium.
3
Analyze
or extract the cells one or two days after the transfection. You can control
the transfection efficiency of GFP-transfected cells by fluorescence microscopy
or flow cytometry.
Conditions (for one well of a 6-well plate = for 10 cm2)
|
Cell type |
Medium |
DNA |
Plus-R. |
Lipofectamine |
Incubation |
efficiency |
|
HeLa |
DMEM |
1.5 µg |
5 µl |
3 µl |
7 h |
50 - 70 % |
|
293 |
DMEM |
1 µg |
4 µl |
3 µl |
3 - 5 h |
70 - 100 % |
|
CHO |
MEM-alpha |
1 µg |
5 µl |
2 µl |
3.5 h |
50 - 70 % |
|
HUVEC |
M 199 |
1.5 µg |
8 µl |
4 µl |
2 - 3 h |
10 - 20 % |