Molecular Techniques and Methods

High Efficiency Calcium Phosphate-Mediated Transformation

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

This is a highly efficient method to obtain stable transformation of mammalian cells with supercoiled plasmid DNAs. The calcium phosphate-DNA coprecipitate is allowed to form in the tissue culture medium during prolonged incubation (15-24 hours) under controlled conditions of pH (6.96) and CO2 tension (2-4%). It should be noted that linear DNAs yield very low transformation frequencies using this protocol, perhaps because the slow formation of the calcium phosphate-DNA coprecipitate delays protection of the DNA from nucleases. The best results have been obtained using supercoiled plasmid DNAs purified by two rounds of equilibrium centrifugation in CsCl-ethidium bromide density gradients.




MATERIALS AND SOLUTIONS

2 x BES-buffered Saline (100 ml)
50 mM BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid) ----- 1.07 g
280 mM NaCl ----------------------------------------------------------- 5.6 ml of 5 M NaCl
1.5 mM Na2HPO4.2H2O ------------------------------------------------ 0.027 g
Add distilled H2O to make a final volume of ---------------------------- 100 ml
  • Adjust the pH to 6.96 with conc. HCl.
  • Sterilize the solution by passage through a 0.22 um filter.
  • Store in aliquots at -20oC.


    2.5 M CaCl2
    CaCl2.2H2O ------------------------------------------------------------ 7.35 g
    Add distilled H2O 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.




    PROCEDURES

    1. Harvest exponentially growing cells by trypsinization.

    2. Replate cells at a density of 5 x 105 cells/cm2 in 90-mm tissue culture dishes in the serum-containing complete medium (10 ml).

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

    4.
    DNA Preparation
  • Mix 20-30 ug of superhelical plasmid DNA with 0.5 ml of 0.25 M CaCl2.
  • Add 0.5 ml of 2 x BES-buffered Saline.
  • Incubate the mixture for 10-20 minutes at room temperature.
  • Do not expect a visible precipitate to form during this time.



  • 5. Add the CaCl2/ DNA/ BES-buffered Saline solution dropwise to the dishes of cells, swirling gently to mix well.

    6. Incubate the cultures for 15-24 hours at 37oC in a humidified incubator in an atmosphere of 2-4% CO2.
  • The calcium phosphate-DNA complex forms slowly in the medium under conditions of low pH and precipitates gradually onto the cells during the incubation in an atmosphere containing low concentrations of CO2.
  • The nature of the precipitate is affected by the amount of DNA used.
  • A transition from a coarse precipitate to a fine precipitate occurs at the optimal DNA concentration (2-3 ug/ml in the growth medium).


  • 7. Remove the medium by aspiration, and rinse the cells twice with medium.

    8. Add 10 ml of fresh medium, and incubate the cultures for 24 hours at 37oC in a humidified incubator in an atmosphere of 5% CO2.

    9. Following 18-24 hours of incubation in nonselective medium to allow expression of the transferred gene(s) to occur, trypsinze and replate the cells in the appropriate selective medium.

    10. Change the medium every 2-4 days for 2-3 weeks to remove the debris of dead cells and to allow colonies of resistant cells to grow.

    11. The dilution at which the cells should be replated to yield well-separated colonies will be determined by the efficiency of stable transformation, which can vary over several orders of magnitude.
  • The efficiency is dependent on (1) the recipient cell type, (2) the nature of the introduced gene and the efficacy of the transcriptional control signals associated with it, and (3) the amount of donor DNA used in the transfection.


  • 12. Individual colonies may be cloned and propagated for assay.

    13. To get a permanent record of the numbers of colonies, fix the remaining cells with ice-cold methanol for 15 minutes.
  • Stain the cells with 10% Giemsa for 15 minutes at room temperature before rinsing in tap water.


    NOTES


    KIT INFORMATION


    REFERENCES


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

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