Molecular Techniques and Methods

Isolation of Genomic DNA from Mammalian Cells

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION



MATERIALS AND SOLUTIONS

Cell Resuspension Buffer (10 ml)
10 mM Tris-HClpH8.0 ------------------------------ 100 ul of 1 M Tris-HClpH8.0
10 mM EDTA --------------------------------------- 100 ul of 0.5 M EDTA
Add deionized H2O to make a final volume of --- 10 ml
  • Keep at 4oC.


    Proteinase K Solution (1 ug/ul)
    Proteinase K ---------------------------------------- 1 mg
    Distilled H2O --------------------------------------- 1 ml
  • Store at -20oC.



    PROCEDURES

    1. Trypsinize, harvest and resuspend cells at 104 cells/ ul in 200 ul Cell Resuspension Buffer.

    2. To cell resuspension, add 10 ul SDS and 20 ul Proteinase K to a final concentration of 0.5% and 200 µg/ml, respectively.

    3. Mix and incubate at 55°C for 2 hours.

    4. Add 10 ul, 5 M NaCl to a final concentration of 0.2M.

    5. Extract with equal volumes of phenol:chloroform (1:1):IAA.

    6. Extract with chloroform:IAA.

    7. To evaporate the chloroform, use vaccum dryer for 5 min.

    8. Add 10 ul, RNase A (DNase-free and protease-free) to a final conc. of 25 µg/ml and incubate for 1 hour at 37°C.
  • The concentration of the enzyme may vary for different cell types.

    9. Extract with phenol:chloroform (1:1):IAA.

    10. Extract with chloroform:IAA.

    11. Precipitate DNA by adding 1.5 vol. (375 ul) of 100% Ethanol.

    12. Centrifuge briefly to pellet the DNA.

    13. Resuspend the pellet in TE buffer or H2O.



    NOTES

  • Do not over-dry the DNA pellet. It will be very difficult to dissolve the DNA.



    REFERENCES

  • Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1.31-1.38, 2001.
  • Sharma, R.C., et al., A rapid procedure for isolation of RNA-free genomic DNA from mammalian cells, BioTechniques, 14, 176-178, 1993.





  • Please send your comment on this protocol to "editor@MolecularInfo.com".
  • 4/16/2018

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