View source for Transfection of HEK293T cells

{| class="wikitable"
!Materials
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|Plasmid DNA
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|2x HBS- buffer, pH 7.05 (correct pH is extremely critical !!)
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|2.5 M CaCl2 (sterile)
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|25 mM chloroquin (sterile)
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'''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