Generic Transfection Protocol
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Use this procedure to transfect plasmid DNA into HEK 293 cells in a 24-well format (for other formats, see Table 2 below). All amounts and volumes are given on a per well basis.
- The day before transfection, trypsinize and count the cells. Plate 0.5 -1.25x105 cells per well in 0.5 ml of complete growth medium. Cell density should be 50-80% confluent on the day of transfection. See Table 1 for other culture vessels.
- (Optional) The day of transfection, remove growth medium from cells and replace with 0.5 ml of complete growth medium.
- For each well of cells to be transfected, dilute 0.5 μg of DNA in 25 μl of DMEM
- For each well of cells, dilute 1.5 μl of GeneExpresso Max Transfection Reagent in 25 μl of DMEM.
- Mix above diluted solutions, mix gently and incubate 20 minutes at room temperature to form DNA- Transfection Reagent complexes.
- After 20 minute incubation, add 50 μl of the DNA- Transfection Reagent complexes directly to each well containing cells and mix gently by rocking the plate back and forth.
- Complexes do not have to be removed following transfection. Incubate the cells at 37oC in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene expression.
Table 1. Recommended number of cells to seed the day before transfection.
| Culture vessel | Surface area per well (cm2) |
Number of adherent cells to seed |
Volume of medium per well to seed the cells |
| 96-well |
0.3 |
25,000 | 100 μl |
| 48-well |
1 |
75,000 |
200 μl |
| 24-well |
2 |
125,000 |
500 μl |
| 12-well |
4 |
250,000 |
1 ml |
| 6-well / 35 mm |
10 |
625,000 |
2 ml |
| 60 mm / flask T25 cm2 |
25 |
250,000 - 800,000 |
4~5 ml |
| 100 mm / flask T75 cm2 |
75 |
1 x 106 - 2 x 106 |
8~10 ml |
| 150 mm / flask T175 cm2 |
175 |
2 x 106 - 5 x 106 |
20~30 ml |
Table 2. Recommended Amounts for Different Culture Vessel.
| Culture Dish/Plate |
Culture Medium |
Plasmid DNA |
Volume dilution medium |
Transfection Reagent GEM |
| 96 well plate |
100 μl |
100 ng |
2 X 10 μl |
0.3 μl |
| 48 well plate |
200 μl |
200 ng |
2 X 20 μl |
0.6 μl |
| 24 well plate |
500 μl |
500 ng |
2 X 25 μl |
1.5 μl |
| 12 well plate |
1 ml |
1 μg |
2 X 50 μl |
3 μl |
| 6 well plate |
2 ml |
2.5 μg |
2 X 150 μl |
7.5 μl |
| 60 mm dish |
5 ml |
6.5 μg |
2 X 375 μl |
20 μl |
| 10 cm dish or T75 flask |
8 ml |
10 μg |
2 X 500 μl |
30 μl |
| 15 cm dish or T175 flask |
20~ 25 ml |
25~30 μg |
2 X 1.5 ml |
75 μl ~90 μl |