1. Cell line to be transfected
2. Cell culture medium
3. H₅₀N₂₈₀ (50 mM Hepes / 280 mM NaCl pH=7.1)
4. P₇₀ (70 mM sodium phosphate pH=7.1)
5. 2 M CaCl₂
   (All solutions are sterilized by autoclaving. Store at RT)
6. High molecular weight calf thymus carrier DNA (Boehringer Mannheim)
   If not commercially available, use lab made genomic DNA instead.
7. Tissue culture dishes
8. 50ml sterile Falcon tube
9. Serological pipettes (sterile)
10. Sterile H2O
11. pH meter
12. Bubbling apparatus
13. Selection drug

1. Plate 1.3 X 10⁵ RK3E cells in DMEM containing 10% FBS using 10 cm tissue culture dishes one day prior to transfection.
2. Before transfection, make HNP buffer by mixing 49 ml H₅₀N₂₈₀ and 1ml P₇₀ in 50 ml sterile tube. (check again the pH between 7.05 and 7.15, the pH is very critical for transfection efficiency) and 0.5 M CaCl_2.
3. For each tissue culture dish dissolve 40 ug carrier DNA and 10ug plasmid DNA in 0.5 ml dH2O. Add 0.5 ml 0.5M CaCl2 and mix gently.
4. Add 1ml of DNA-CaCl2 solution slowly to 1 ml HNP buffer in 50ml Falcon tube while gently bubbling the latter.
5. Let the precipitate develop for 30-60 min at RT.
6. Resuspend precipitate by gentle pipetting and disperse over the dish. Swirl gently.
7. Incubate undisturbed for 16-22 hr at 37^oC and 5% CO2.
8. Swirl plates gently and remove transfection medium completely, add 10 ml of DMEM containing 10% FBS. Maintain cells by changing medium twice a week. Biological phenotype of transforming genes will appear about 7-10 days following transfection.
9. For marker selection, change medium after transfection to DMEM containing 10% FB serum and the selective drug. selection reqirements vary dependent on the marker, but selection will be ordinarily completed after 10-14 days.
    
    

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(Refer to Kraus, M.H. et al. PNAS 81: 5384-5388, 1984)