1 mg/ml DEAE Dextran 100 mg DEAE Dextran

(MW 5x10⁵)

up to 100 ml with sterile1X TBS (10 mM Tris 7.5, 50 mM NaCl)

50 mg/ml chloroquine 500 mg chloroquine

up to 10 ml with sterile 1X PBS

• Split cells 1:2 on the day prior to the transfection.

• Mix DEAE Dextran as follows per 10 cm dish:

500 microliters DME-

500 microliters DEAE Dextran (MW 5x10⁵ 1mg/ml in TBS)

Add 10 micrograms plasmid DNA

• Trypsinize cells at 37° for 5 minutes, wash twice in DME-.

• Resuspend the cells in the DNA mixture (1 ml) and plate on a 10 cm dish, incubate in the tissue culture incubator for 30 minutes.

• Some cells will stick to the plate and some will not. Rinse the plate with DME- to get the loose cells and pellet. Resuspend in 10 ml DME+10% FCS and add to the original plate with the cells that were adherent. Return to the tissue culture incubator.

• 2-3 days later, harvest the cells by washing in PBS and then incubating in PBS + 2mM EDTA at 37° for 10 minutes. Alternately, the cells can be scraped off the plate.

• Proceed according to desired purification/analytical protocol.