Calcium phosphate precipitation

From Karl Matter

                                                                                                            9.9.99

 

References : Wigler et al. (1979) PNAS 76,1373-1376.

 

 

1.    Mix fresh 2x HEPES buffer (280 mM NaCl , 50 mM HEPES, 1.5 mM NaPO4, pH  7.1); filter sterilize

2.    Mix fresh 0.25 M CaCl2 from 1M stock and filter sterilize

3.    Test precipitate formation by adding 0.25 ml CaCl2 dropwise to 0.25 ml 2xHEPES on vortex. Note cloudyness that forms after a few minutes.

4.    Add 20 to 50 ”g DNA to 1 ml 2x HEPES buffer

5.    Add 1 ml 0.25 M CaCl2 dropwise shaking vigorously after each drop (keep tube on vortex all the time)

6.    Allow precipitate to form 30 minutes at room temperature.

7.    Vortex and add to 10 cm cell culture plates (~ 30 confluent; split the day before) containing 3ml medium + FBS (prewarmed).

8.    Incubate 2-4 hours or over night at 37ĄC

For MDCK:    - add precipitate to cells without medium and incubate for 20 to 30 min at 37ĄC (shake once after about 10 min)

                        - add 7 ml of warm medium and incubate over night

9. Remove media. Glycerol shock with 12.5 % sterile glycerol in medium containing 10 mM Hepes, pH7.4. Incubate 1 to 2 min at room temperature

10.  Remove glycerol solution and carefully rinse cells two times with PBS.

11.  Add medium with serum.

12.  Start selection 48 hours later (use O.5 mg/ml G418 if the plasmid carries a neomycin resistance gene).  Make different dilutions of the cells in 15cm dishes. 

Cell will grow apparently normally for a couple of days and then start to die.  After approximately 2 weeks you will start to see colonies.

 

For 6-well plates:

- add 15 to 25 ”g DNA to 0.5 ml 2xHEPES buffer and precipitate with 0.5 ml 0.25 M CaCl2 dropwise on Vortex. 

- allow precipitate to form 30 minutes at room temperature

- add 1 ml suspension to one well

- incubate for 10 min in incubator (MDCK: 30 min)

- add 2.5 ml normal medium and incubate 3 to 6 hours or over night at 37ĄC (MDCK: overnight)

- Remove media. Glycerol shock with 12.5 % sterile glycerol in medium containing 10 mM Hepes, pH7.4

 

 

Reagents:

 

2x HEPES BUFFER

0.5 M HEPES pH 7.1                     5 ml                                 667 ”l

3.0 M NaCl                                        4.65 ml                            623 ”l

1.0 M NaPO4 pH 7.1                      75 ”l                                10 ”l

dH2O to volume                               50 ml                               6.67 ml

 

Filter sterilize. Make fresh for each use.