Calcium phosphate-mediated transfection into HEK 293T cells

1. IMDM 

2. DMEM complete media
   

3. FBS

4. Pen/Strep

5. HEPES

6. NaCl

7. Na₂HPO₄

8. CaCl₂

9. TRIS-HCl pH 8.0

10. EDTA pH 8.0

11. IMDM media - see recipes

12. 2xHBS buffer - see recipes

13. 2.5M CaCl₂ - see recipes
    

14. 0.1x TE buffer  - see recipes
    

1. Biological hood

2. Pipetors, pipet aid

3. Tips, pipetts

4. 0.22μm filters

5. 15ml tubes

6. 10cm tissue culture dish

7. tissue culture incubator

8. 
   

1.      Seed ~4.5 x 10⁶ cells onto a 10 cm dish a day before transfection.  Use low passage cells (not more than P15).  Do not allow cells to grow to confluence.

2.      Remove media and replace with IMDM media for at least 2 hrs.

3.      Mix plasmids with 0.1x TE to a final volume of 450 ul in 15ml tubes.

4.      Add 50 ul of 2.5 M CaCl₂ to the plasmid mix.

5.      Allow to mix at RT for 5 minutes.

6.      Apply 500 ul of 2x HBS  dropwise to the plasmid mix while vortexing at full speed.

7.      Apply the precipitate to the 293T cells, dropwise around the entire dish.

8.      Incubate 293T cells O/N (14-16 hrs).

9.      Replace media with fresh DMEM complete media.

IMDM   media

IMDM (Iscove)  44.75 ml

FBS                              5

Pen/Strep                      0.25

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Total                            50 ml

2x HBS (store at -20 or -80 ^oC)

281 mM NaCl

100 mM HEPES

1.5 mM Na₂HPO₄, pH 7.12

Filter sterilize (0.22 um)

CaCl₂ (store at -20 ^oC)

2.5 M CaCl₂

Filter sterilize (0.22 um)

0.1x TE buffer (store at 4 ^oC)

10 mM Tris (pH 8.0)

1 mM EDTA (pH 8.0)

Dilute 1:10 with ddH₂O

Filter sterilize (0.22 um)