Transfection protocol for 6-well dish
Note: 1) one 6-well dish for each construct tested, three untreated (DMSO) and
three treated (GSE).
2) include one 6-well dish for untransfected control
- Seed CHO stable cells at 5x104/well the day before transfection.
- Combine tested reporter construct at 1ug /well with 100ul/well serum-free medium
- Add 5ul/well Superfect (Qiagen) to the DNA/medium mixture. Vortex for 10 seconds
- Incubate at RT for 10 min
- Aspirate medium and wash cells once with PBS
- Add 600ul regular growth medium (with FBS) to the mixture from step 3. Mix by pipetting up and down 2 times
- Aspirate PBS and add total 700ul to each well.
- Incubate at 37 oC incubator for 4-6 hrs
- Aspirate medium
- (Optional) wash once with PBS
- Add 4ml regular growth medium to each well
- Add either 4ul DMSO or 4ul GSE (final concentration of 15uM/ml) to wells accordingly
- After 48 hrs, harvest cells for functional assay
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