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Calcium phosphate-mediated transfection into HEK 293T cells

Author: Gal Haimovich, updated date: , view: 29, Q&A: 0
Tags: Cell Culture, Transfection and HEK293T

Materials and Reagents

  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

Equipment

  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


Procedure

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.


Recipes

IMDM   media

 

IMDM (Iscove)  44.75 ml

FBS                              5

Pen/Strep                      0.25

-----------------------------------------

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)

 


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