Transfection of fibroblasts

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.