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The Laboratory of J. Michael Bishop at the University of California, San Francisco
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1. Plate cells to be transformed onto polylysine-coated dishes (see Hint #1) at cell densities of 1 to 2 million per 60 mm dish, 3 to 4 million per 100 mm dish, or 6 to 8 X 106 per 150 mm dish.
2. Aliquot DNA into one-half the appropriate transfection volume (see below) of DMEM without Serum. Aliquot lipofectin into a separate tube of DMEM (also one-half the final volume) at 16 μl/ml (after combining the two solutions, the final lipofectin concentration will be 8 μl/ml).
Transfection media volumes to use:
2 ml for a 60 mm plate
4 ml for a 100 mm plate
5 ml for a 150 mm plate.
Use DNA at a final concentration of 10 μg/ml in transfection media (although a wide range of DNA concentration is effective).
3. Rinse plates with Serum-free DMEM and remove all liquid from the plate.
4. Combine the contents of the DMEM plus DNA tube with the DMEM plus lipofectin tube. Drip this transfection media gently onto the edge of the plate and spread liquid over plate evenly.
5. Incubate in a cell culture incubator at 37°C, 12% CO2 in air for 1 to 5 hr.
6. Without removing the lipofection media, gently add an equal volume of regular media containing 5% Horse Serum/5% Supplemented Calf Serum.
7. For transient transfections, assay for transfected product 2 to 3 days later while for stable transfections, begin G418 selection 3 days later.
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| DMEM with Serum |
| 5% (v/v) Supplemented Calf Serum
DMEM (Dulbecco's Modified Eagle's Medium)
5% (v/v) Horse Serum
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Serum, Calf
Polylysine
Serum, Horse
DMEM
Lipofectin
G-418 (Geneticin)
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1. The polylysine is necessary for PC12 cells to remain adherent to the plate. Polylysine coating may not be necessary with more readily-adherent cell lines.
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