Calcium phosphate transfection protocol2

1. Detech cells with 0.05% tryp­sin–eth­yl­ene­di­amine­tet­ra­ace­tic acid (EDTA) treat­ment.

2. Stop trys­i­ni­za­tion by DMEM–10% FCS.
3. Remove trysin by cen­tri­fu­ga­tion.

4. Make Ca-P complex solu­tion using 2.2 M CaCl2 and 2X Hepes-buffered solu­tion [HBS] with ODN/siR­NA (200–250 nM).

5. Add 70 ul of –cal­cium phos­phate pre­cip­i­tate to 2.5 X 10^5 cell sus­pension

6. Incubate for 25 min with gen­tle shak­ing every 5 min.

7. Add 1 ml of 10% FCS–DMEM,  plate cells in the serum-con­tain­ing medium for 3 to 4 h at 37 °C for adher­ence.

8. Remove medium, wash cells with PBS, add complete medium