Steven Finkbeiner, Departments of Neurology and Physiology, UCSF
http://gweb1.ucsf.edu/labs/finkbeiner/Protocol/protocol_list/pc12_lipofe_transf.shtml
Cells should be plated on collagen-coated dishes, as for normal propagation. If there appears to be large scale loss of cells after Lipofectamine treatment, then it might pay to try using polylysine-coated plates.
- Plate 2 X 106 cells per 60 mm tissue culture dish in DMEM/10% horse serum/5% Fetal calf serum/pen-strep (normal PC12 growth medium) and grow overnight in 10% CO2 incubator. The cells should be 50-70% confluent.
- Remove medium from cells, rinse 1X with 3 ml prewarmed OptiMEM serum-free medium (GIBCO) and add 5 ml OptiMEM and put the cells back in the incubator until ready to transfect.
Transfection
- For each dish of cells aliquot 2 micrograms of the plasmid or plasmid mixture into a 5 ml sterile tube. Add 100 microliters of OptiMEM medium.
- In a separate tube aliquot 15 microliters of 2 mg/ml Lipofectamine (GIBCO) and add 100 microliters OptiMEM.
- Combine the diluted DNA and diluted Lipofectamine, mix gently, and let incubate at room temperature for 15-40 minutes. This allows the DNA and the lipofectamine to form the complexes that will get the DNA into the cells.
- To each transfection mixture add 2.8 ml prewarmed OptiMEM.
- Aspirate the medium from the dish of cells, add the diluted transformation mix, and incubate the cells with the mix in a 10% CO2 incubator for approximately 5 hours.
- At the end of the incubation, remove the transformation mixture and add 5 ml of prewarmed normal PC12 growth medium with serum. Return the cells to the 10% CO2 incubator.
This protocol typical has given 1-2% transfected cells, as judged by staining with X-gal for a CMV-beta-galactosidase reporter gene. I usually let the cells grow for 40 to 60 hours before harvesting in transient transfections.
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