Molecular Techniques and Methods

Isolation of High Molecular Weight DNA from Animal Cells

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Cell Lysis Buffer (1 liter)
320 mM Sucrose ------------------------------- 109.6 g
1% Triton X-100 ------------------------------- 20 ml of 50% Triton X-100
5 mM MgCl2 ----------------------------------- 5 ml of 1 M MgCl2
10 mM Tris-HCl (pH7.6) ----------------------- 10 ml of 1 M Tris-HCl
Add deionized H2O to make a final volume of --- 1 liter
  • Keep at 4oC.


    Saline-EDTA (100 ml)
    25 mM EDTA (pH8.0) ------------------------- 5 ml of 0.5 M EDTA
    75 mM NaCl ----------------------------------- 1.5 ml of 5 M NaCl
    Deionized H2O --------------------------------- 93.5 ml


    Proteinase K Solution (10 mg/ml)
    Proteinase K ------------------------------------ 1 mg
    Distilled H2O ------------------------------------ 1 ml
  • Store at -20oC.




    PROCEDURES

    1. Collect animal cells by centrifugation at 1,500g for 10 minutes at 4oC.

    2. Resuspend the cell pellet at a concentration of 108 cells/ml of cold Cell Lysis Buffer.

    3. Homogenize in a Potter homogenizer with a loose fitting pestle.

    4. Centrifuge at 2,500g for 20 min at 4oC to pellet the nuclei.

    5. Resuspend the pellet in 8 ml (or, 1/10th volume of Cell Lysis Buffer from step 2) of Saline-EDTA,
    and add 0.8 ml of 10% SDS.

    6. Mix briefly by vortexing.

    7. Add 50 ul of the Proteinase K solution (10 mg/ml).

    8. Incubate at 37oC for 3-5 h.

    9. Add 0.5 ml of 5 M sodium percholate and 8 ml of phenol: chloroform: IAA.

    10. Mix gently by inverting tubes for 1 minute.

    11. Centrifuge at 13,000g for 10 min at 10oC.

    12. Collect the aqueous phase with a wide-bore pipet.

    13. Extract with an equal volume of chloroform: IAA.

    14. Mix gently by inverting tubes for 1 minute.

    15. Centrifuge at 13,000 g for 10 min at 10oC.

    16. Collect the aqueous phase with a wide-bore pipet.

    17. Add 2 vol of 100% ethanol to precipitate the DNA.

    18. Spool out the DNA with the glass rod.

    19. Dissolve in 1 ml of TE buffer.

    20. Add 100 ul of 20 X SSC and 10 ul of DNase-free Ribonuclease.

    21. Incubate for 1 h at 37oC.

    22. Extract the solution two times with chloroform: IAA.

    23. Precipitate the DNA by adding 2 volume of 100% ethanol.

    24. Centrifuge at 5,000g for 5 min at 4oC.

    25. Wash the DNA pellet with 70% ethanol.

    26. Air dry briefly.

    27. Dissolve the DNA in 200 ul of TE buffer.




    NOTES

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





  • KIT INFORMATION




    REFERENCES



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

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