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Protocol

Lipofectin Transfection Protocol
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Lipofectin is a synthetic lipid polymer which is able to fuse with the membranes of cells in physiological salt solutions. This fusion somehow allows the efficient entrance of DNA into the cells. This method can be used to produce either stable transfectants or transient transfectants. The major points of the method are: 1. simplicity no tricky buffers, aeration steps, etc. 2. Efficiency I have found it more efficient thant CaPO4 or DEAE-Dextran for COS cells. 3. Reproducibility The weak points are: 1. Expense - its costs approximately $5 per standard 100 mm plate, which can add up if you are doing lots of assays. 2. Serum-free media - the transfection must occur in the absence of serum. If your cells can not tolerate this for any length of time this will limit the efficiency of the transfection. There are multiple protocols for doing the transfection. This is the one I found to work the best while still being fairly simple.
Protocol: - for a standard 100 mm plate 1. Plate calls in their normal growth media so that they will be 80-90% confluent for the transfection. I normally plate them 16-20 hours before the transfection. 2. Rinse the cells three times with 3 mls. of Optimem to remove all traces of serum. 3. Add 2.5 mls of optimem containing the plasmid DNA to each dish (see note below). 4. Add 2.5 mls of optimem containing the lipofectin (made up in a polystyrene tube - not polypropylene) to the plate. 5. Rock gently back and forth to mix the DNA and lipofectin. 6. Incubate for 6-20 hours. The longer the better if the cells can tolerate being out of serum for that long. 7. Add 10 mls of media with serum to stop the reaction. If you grow the cells in serum f ree media you can wash the cells twice to stop the reaction. Incubate for 24-60 hours and then assay as you normally would.
Lipofectin Transfection Protocol

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