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MARTIN MARX
sure not to use fetal calf serum because this will detach the cells immediately! Y ou will need two flasks of cells: one for the transfection, the
other as a control.
3. Grow the cells until about 75-90% confluent.
4. Prepare a stock solution ofPolybrene (30 mg/ml) in PBS, filter through
a 0.2 )lm filter.
5. Resuspend the ethanol-precipitated DNA in 50 )ll of sterile water orTE.
Use all DNA recovered from the miniprep.
6. Add the DNA aseptically to 4 ml complete medium. Add polybrene to
the medium to a final concentration of 30 )lg/ml and mix.
7. Remave the old medium from the cells in the 75 cm 2 flask, and replace
with the DNA-polybrene containing medium. 4 ml should be sufficient
to just cover the celllayer in a 75 cm 2 flask. Allow contact with the cells
for 6-8 hours at 37°C in a humidified atmosphere. Rock the flask occasionally.
8. Remave the old medium by aspiration.
9. Add 4 ml of complete medium containing 20% DMSO. This medium
should be used at room temperature, not at 37°C! Gently rock the flask
two or three times and allow the DMSO to act on the cells for exactly 4
minutes.
10. Wash the cells three times with 10 ml complete medium. Feed with 10
ml complete medium and return flasks to the incubator.
Note: There is a small chance that the cells may detach during the DMSO
shock procedure. Forthis reason, the flask should be handled with extreme
care. For the DMSO shock and subsequent washes, avoid tauehingor rinsing the monolayer directly. Add the DMSO medium to the ceiling of the
inverted flask, then invert the flask slowly and carefully. W ash the cells the
same way.
Selection of successfully transfected \jf2 cells with G418
Since pLXSN carries the neomycin resistance gene, succesfully transfected
cells can be easily selected for in medium supplemented with the toxic aminoglycoside geneticin ( G418).
MARTIN MARX
sure not to use fetal calf serum because this will detach the cells immediately! Y ou will need two flasks of cells: one for the transfection, the
other as a control.
3. Grow the cells until about 75-90% confluent.
4. Prepare a stock solution ofPolybrene (30 mg/ml) in PBS, filter through
a 0.2 )lm filter.
5. Resuspend the ethanol-precipitated DNA in 50 )ll of sterile water orTE.
Use all DNA recovered from the miniprep.
6. Add the DNA aseptically to 4 ml complete medium. Add polybrene to
the medium to a final concentration of 30 )lg/ml and mix.
7. Remave the old medium from the cells in the 75 cm 2 flask, and replace
with the DNA-polybrene containing medium. 4 ml should be sufficient
to just cover the celllayer in a 75 cm 2 flask. Allow contact with the cells
for 6-8 hours at 37°C in a humidified atmosphere. Rock the flask occasionally.
8. Remave the old medium by aspiration.
9. Add 4 ml of complete medium containing 20% DMSO. This medium
should be used at room temperature, not at 37°C! Gently rock the flask
two or three times and allow the DMSO to act on the cells for exactly 4
minutes.
10. Wash the cells three times with 10 ml complete medium. Feed with 10
ml complete medium and return flasks to the incubator.
Note: There is a small chance that the cells may detach during the DMSO
shock procedure. Forthis reason, the flask should be handled with extreme
care. For the DMSO shock and subsequent washes, avoid tauehingor rinsing the monolayer directly. Add the DMSO medium to the ceiling of the
inverted flask, then invert the flask slowly and carefully. W ash the cells the
same way.
Selection of successfully transfected \jf2 cells with G418
Since pLXSN carries the neomycin resistance gene, succesfully transfected
cells can be easily selected for in medium supplemented with the toxic aminoglycoside geneticin ( G418).