Transfection of fibroblasts: Difference between revisions
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Created page with "=== Materials === Plasmid DNA OptiMEM Lipofectamin 3000 or jetPrime === Procedure === The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x10^4 for 24 well, 1.5x10^5 for 6 well plate) Change medium to remove and dead cells and debris Which transfection reagent works best for your cells has to be determined empirically: ==== '''JetPrime Protocol''' for..." |
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Plasmid DNA | |+ | ||
!Materials | |||
OptiMEM | |- | ||
|Plasmid DNA | |||
Lipofectamin 3000 or jetPrime | |- | ||
|OptiMEM | |||
|- | |||
|Lipofectamin 3000 or jetPrime | |||
|} | |||
=== Procedure === | === Procedure === | ||
The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x10 | The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x10<sup>4</sup> for 24 well, 1.5x10<sup>5</sup> for 6 well plate) | ||
Change medium to remove and dead cells and debris | Change medium to remove and dead cells and debris | ||
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* ''Optional'': Medium exchange after 4h | * ''Optional'': Medium exchange after 4h | ||
Incubate cells at 37° C and 5% CO2 | Incubate cells at 37° C and 5% CO2. | ||
Analyze after 24-72h. | Analyze after 24-72h. | ||
Lipofectamin:DNA ratio might be varied to increase transfection efficiency. | Lipofectamin:DNA ratio might be varied to increase transfection efficiency. | ||
Latest revision as of 17:52, 15 February 2025
| Materials |
|---|
| Plasmid DNA |
| OptiMEM |
| Lipofectamin 3000 or jetPrime |
Procedure
The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x104 for 24 well, 1.5x105 for 6 well plate)
Change medium to remove and dead cells and debris
Which transfection reagent works best for your cells has to be determined empirically:
JetPrime Protocol for 6 well plate (volumes for 24 well in brackets)
- Dilute 2.5 µg (0.5 µg) DNA in 200 µl (50 µl) jetprime Buffer W
- Vortex for 10 sec
- Spin down
- Add 5 µl (1 µl) of jetPrime reagent
- Vortex for 10 sec
- Spin down
- Incubate 10 min at room temperature
- Add transfection mix dropwise to the cells
- Optional: Medium exchange after 6-8h
Lipofectamin protocol for 6 well (volumes for 24 well in brackets)
- Dilute 5 µl (1 µl) Lipofectamin 3000 reagent in 125 µl (25 µl) OptiMEM
- Vortex and spin down
- Dilute 2.5 µg (1µg) DNA + 5 µl (2 µl) P3000 Reagent in 125 µl (25 µl) OptiMEM
- Vortex and spin down
- Add diluted DNA to diluted Lipofectamin
- Vortex and spin down
- Incubate 10-15 min at room temperature
- Add 250 µl transfection mix dropwise to the cells
- Optional: Medium exchange after 4h
Incubate cells at 37° C and 5% CO2. Analyze after 24-72h.
Lipofectamin:DNA ratio might be varied to increase transfection efficiency.