Transfection of HEK293T cells

Materials
	Plasmid DNA
	2x HBS- buffer, pH 7.05 (correct pH is extremely critical !!)
	2.5 M CaCl2 (sterile)
	25 mM chloroquin (sterile)

Procedure

• 1 day before transfection: cells split 1:3 up to 1:5 in 10cm dishes

• Next day: Set up for each 10cm dish: 500 μl ddH2O (sterile) in 14 ml PP Greiner tube

• Add 5 μg (or adapted amounts) of plasmid DNA

• Add 500 μl 2x HBS buffer, mix sample

• Add drop-wise and during constant mixing on vortex 50 μl sterile CaCl2 to sample

• Incubate for 5 min at room temperature to allow DNA/Ca2+ precipitate formation

• During DNA/Ca2+ precipitate formation, add 10 μl of chloroquin to cells in 10 cm dish

• Add drop-wise 1 ml of DNA/Ca2+ precipitate sample on cells in 10 cm dish (“carpet bombing”), be carefull not to detach cells

• Incubate cells for 6- 8 h in incubator

• Remove medium and add 8 ml/dish of fresh medium carefully onto cells

• Expression of transgenes is usually highest on day 2 after the transfection