Molecular Techniques and Methods

Calcium Phosphate-Mediated Transformation

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The uptake of DNA by cells in culture is markedly enhanced when the nucleic acid is presented as a calcium phosphate-DNA coprecipitate.



MATERIALS AND SOLUTIONS

2 x HBS (HEPES-Buffered Saline) (100 ml)
280 mM NaCl ------------------------------------- 5.6 ml of 5 M NaCl
10 mM KCl ---------------------------------------- 1 ml of 1 M KCl
1.5 mM Na2HP04.2H20 ---------------------------- 0.027 g
12 mM Dextrose ------------------------------------ 0.2 g
50 mM HEPES ------------------------------------- 5 ml of 1 M HEPES
Add deionized H2O to make a final volume of ----- 100 ml
  • Adjust the pH to 7.05 with 0.5 N NaOH.
  • Sterilize the solution by passage through a 0.22 um filter.
  • Store in 5 ml aliquots at -20oC.


    2.5 M CaCl2 (20 ml)
    CaCl2.2H2O ------------------------------------------- 7.35 g
    Add distilled H20 to make a final volume of --------- 20 ml
  • Sterilize the solution by passage through a 0.22 um filter.
  • Store in 1 ml aliquots at -20oC.


    0.1 x TE (pH 8.0) (100 ml)
    1 mM Tris-HCl (pH 8.0) ---------------------------- 0.1 ml of 1 M Tris-HCl
    0.1 mM EDTA (pH 8.0) ---------------------------- 20 ul of 0.5 M EDTA
    Distilled H20 ---------------------------------------- 99.88 ml
  • Sterilize the solution by passage through a 0.22 um filter.
  • Store in aliquots at 4oC.




    PROCEDURES

    1. DNA Preparation
  • Dissolve the DNA (20 ug/106 cells) in 0.1 x TE (pH 8.0) at a concentration of 40 ug/ml.
  • To obtain the highest transformation efficiencies, plasmid DNAs should be purified by equilibrium centrifugation in CsCl-ethidium bromide density gradients.
  • If smaller amounts of DNA are used, carrier DNA should be added to adjust the concentration to 40 ug/ml.
  • Eukaryotic carrier DNA prepared in the laboratory usually gives higher transfection efficiencies than commercially available DNA such as calf thymus or salmon sperm DNA. Carrier DNA should be sterilized before use by extraction with chloroform.


  • 2. Harvest exponentially growing cells by trypsinization.

    3. Replate cells at a density of 1-2 x 105 cells/cm2 in 60-mm tissue culture dishes in the serum-containing medium.

    4. Incubate the cultures for 20-24 hours at 37oC in a humidified incubator in an atmosphere of 5-7% CO2.

    5. For each 60-mm monolayer of cells to be transfected, prepare the calcium phosphate-DNA coprecipitate as follows:
  • Place 220 ul of the DNA prepared in step 1 and 250 ul of 2 x HBS in a disposable, sterile 5 ml plastic tube.
  • Slowly add 24 ul of 2.5 M CaCl2 (with gentle mixing over a period of 30 seconds).
  • Reaction mixtures can be doubled or quadrupled in volume if a larger number of cells are to be transfected. When the volumes are quadrupled, a larger tube (e.g., a 15-ml Falcon 2059 tube) should be used.


  • 6. Incubate the mixture for 20-30 minutes at room temperature, during which time a fine precipitate should form.

    7. At the end of the incubation, pipette the mixture up and down once to resuspend the precipitate.

    8. Transfer the calcium phosphate-DNA suspension into the medium above the cell monolayer.
  • (Use 0.5 ml of suspension for 5 ml of medium in a 60-mm dish.)


  • 9. Rock the dish gently to mix the medium, which will become yellow-orange and turbid.

    10. Incubate the transfected cells for up to 24 hours at 37oC in a humidified incubator in an atmosphere of 5-7% CO2.

    11. Remove the medium and precipitate by aspiration.

    12. Wash the monolayer once with sterile phosphate-buffered saline (PBS), and add 5 ml of prewarmed complete growth medium.

    13. Return the cells to the incubator for 24-60 hours before assaying for transient expression of the transfected DNA or replating the cells in the appropriate selective medium for the isolation of stable transformants.


    14. Transient Expression:
  • Harvest the cells 48-60 hours after transfection for analysis of RNA or DNA by hybridization. Newly synthesized protein may be analyzed by radioimmunoassay, by western blotting, by immunoprecipitation following in vivo metabolic labeling, or by assays of enzymatic activity in cell extracts.
  • For assays that involve replicate samples or treatment of transfected cells under multiple conditions or over a time course, it is desirable to avoid dish-to-dish variation in transfection efficiency. In these cases, it is best to transfect large monolayers of cells (90-mm dishes) and then to trypsinize the cells after 24 hours of incubation and distribute them among several smaller dishes.



  • Stable Transformation:
    Following 18-24 hours of incubation in nonselective medium to allow expression of the transferred gene(s) to occur, the cells are trypsinized and replated in the appropriate selective medium. This medium should be changed every 2-4 days for 2-3 weeks to remove the debris of dead cells and to allow colonies of resistant cells to grow. Individual colonies may be cloned and propagated for assay.




    NOTES

    Published procedures differ widely in the manner and rate of mixing of DNA and CaCl2.
  • Some advise against anything but the gentlest agitation and suggest that air bubbled from an electric pipetting device, should be used to mix the solution.
  • Others advocate continuous slow mixing during addition of the DNA solution, followed by gentle vortexing. The object is to avoid the rapid formation of coarse precipitates that results in a decreased efficiency of transformation.



  • KIT INFORMATION


    REFERENCES

  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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