• Allow cells to attach and grow overnight in incubator

• Calculate volume of maxi-prep DNA that has 10µg DNA. If this volume is less than 100µl then add water to bring volume to 100µl

• Add 1/10 volume of 3M Sodium Acetate and mix

• Add 2.5 volumes of 100% ethanol and mix

• Allow to precipitate at room temperature for at least 15 minutes

• Centrifuge at top speed in Eppendorf centrifuge for at least 20 minutes

• Using sterile procedure in tissue culture hood, carefully decant the supernatant

• In hood, gently wash DNA pellet with 500µl of 75% ethanol

• Allow pellet to dry in hood

• Resuspend DNA in 20µl TE

• Solution A (prepare in a polypropylene tube)
Dilute 2µg of sterile DNA into 100µl Opti-MEM

• Solution B (prepare in a polypropylene tube)
Dilute 15µl LipofectAmine reagent into 100µl Opti-MEM

• Combine Solutions A and B. Mix gently, and allow DNA-liposome complexes to form for 30 minutes at room temperature (15 to 45 minutes is OK)

• While DNA-liposome complexes are forming, aspirate media from cells, and replace with 2ml of Opti-MEM. This washes any serum proteins that could limit transfection efficiency away from the cells

• Add 800µl of Opti-MEM to the tube containing solution A&B

• Aspirate wash media from cells

• Gently overlay the diluted DNA-liposome solution onto cells

• Incubate for 2 to 24 hours. 5 hours is recommended

• Aspirate media, and allow cells to recover overnight with 2ml of 925 with 5% serum

• Replace "recovery" media with 2ml Opti-MEM

• Allow cells to condition media for 36 to 72 hours

• Harvest media and/or cells at the end of conditioning time