( p 3 l reagent 2 g DNA...)Uses and Applications of FuGENE 6 Transfection Reagent

3 l reagent

2 g DNA

6 l reagent

1 g DNA

2. Prepare the complex example given for 6-well plate

Prepare the DNA

• Select a plasmid that you know transfects your cell line, and make sure you are able to measure the expression of reporter gene or transfected protein. Plasmids with reporter genes are available from Roche Applied Science.
• Handle the DNA as aseptically as possible to avoid contaminating your cell cultures during the transfection. Use sterile pipettes, tips, vials, etc.
• Accurate DNA concentrations are important. Determine the concentration of the DNA with a spectrophotometer. Transfection-grade DNA should have a 260/280 ratio of 1.8.
• Check the stock DNA concentration, and determine how much you will need for 1 and 2 g. If the stock is 2 g/l, then decide if you can accurately pipet 0.5 l. It might be better to dilute the DNA using procedures that give accurate dilutions. If you need to dilute the DNA, use sterile DNase-free water or sterile DNase-free TE buffer as the diluent.
• Calculate accordingly for your DNA concentrations (i.e., concentrations of 0.5 g/l work well; for 1 g, measure 2 l, and for 2 g, measure 4 l).

• Remove the FuGENE 6 Reagent from the refrigerator or freezer and allow it to warm to room temperature. Meanwhile, look at the cells microscopically. Make sure they look healthy and are 50-80% confluent. Return the cells to the incubator.
• Warm serum-free DMEM (no additives) to room temperature. Aliquot 94
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