Molecular Techniques and Methods

Isolation of Genomic DNA from Plant Tissues

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

This method is based on the CTAB-Nucleic acid extraction procedures that make it possible to extract purified high molecular weight (> 50 kb) plant DNA. The basis for the separation of polysaccharides from nucleic acids is their differential solubilities in the presence of CTAB. Tissues as small as individual ovules and embryos, or small pieces of tissue from various parts of the same plant, can be used.

  • In order to release the cellular constituents, the cell walls must be broken by grinding the tissue in liquid nitrogen with a mortar and pestle.

  • To release the DNA into the Extraction Buffer, the cell membranes must be disrupted by using a detergent (CTAB; cetyltrimethylammonium bromide).

  • The time between thawing of the frozen, pulverized tissue and its exposure to the Extraction Buffer should be minimized to avoid nucleolytic degradation of the DNA.

  • To protect DNA from the endogenous nucleases, the detergents and also EDTA (ethylenediaminetetraacetic acid) are used. EDTA is a chelating agent that binds magnesium ions, generally considered a necessary cofactor for most nucleases.

  • The buffer/tissue mixture is emulsified with either chloroform or phenol to denature and separate the proteins from the DNA.

  • Shearing of the DNA should be minimized. DNA in solution should be handled very gently. Typically, 50-100 kb DNA can be obtained.





  • MATERIALS AND SOLUTIONS

    2 X Extraction Buffer (100 ml)
    2% CTAB (w/v) ----------------------------------- 2 g
    100 mM Tris-HCl (pH 8.0) ------------------------ 10 ml of 1 M Tris-HCl
    20 mM EDTA (pH 8.0) ---------------------------- 4 ml of 0.5 M EDTA
    1.4 M NaCl ---------------------------------------- 28 ml of 5 M NaCl
    1% PVP (polyvinylpyrrolidone; MW 40,000) ------- 1 g
    Add deionized H2O to make a final volume of ------- 100 ml
  • Warm up to 65oC before use.



  • 10% CTAB (Hexadecyl [or, Cetyl] trimethylammonium bromide) Solution (100 ml)
    10% CTAB ----------------------------------------- 10 g
    0.7 M NaCl ----------------------------------------- 14 ml of 5 M NaCl
    Add deionized H2O to make a final volume of ------- 100 ml


    Precipitation Buffer (100 ml)
    1% CTAB ------------------------------------------- 1 g
    50 mM Tris-HCl (pH 8.0) --------------------------- 5 ml of 1 M Tris-HCl
    10 mM EDTA (pH 8.0) ----------------------------- 2 ml of 0.5 M EDTA
    Add deionized H2O to make a final volume of ------- 100 ml


    High-Salt TE Buffer (100 ml)
    10 mM Tris-HCl (pH 8.0) --------------------------- 1 ml of 1 M Tris-HCl
    1 mM EDTA (pH 8.0) ------------------------------- 0.2 ml of 0.5 M EDTA
    1 M NaCl -------------------------------------------- 20 ml of 5 M NaCl
    Add deionized H2O to make a final volume of -------- 100 ml


    RNase Stock Solution
    1 mg/ml RNase A
    100 U/ml RNase Tl
  • The solution should be heated to boiling in a water bath for 30 min to destroy any DNases.
  • It can be kept frozen until needed.




    PROCEDURES

    1. Grind 0.5 g tissue with the liquid nitrogen in a mortar and pestel (or in a microfuge tube).

    2. Transfer the ground tissue to a microfuge tube.

    3. Add 0.5 ml of 65oC-warmed 2 X Extraction Buffer.
  • If a low yield is expected add about 20 ug of yeast tRNA as a carrier to aid the precipitation in later steps.

    4. Add one volume of chloroform/ isoamyl alcohol (24:1).

    5. Mix gently to form an emulsion.

    6. Centrifuge in a microfuge for 1 minutes at 12,000g.

    7. Transfer the supernatant solution from the top (aqueous) phase to a new microfuge tube. Discard lower (chloroform) phase.

    8. Add 1/10th volume of the 10% CTAB Solution and mix.

    9. Perform another chloroform/ isoamyl alcohol (24:1) extraction as in steps 4-7.

    10. Add an equal volume of Precipitation Buffer and mix gently.

    11. Centrifuge for 30 seconds at 7,500g. Discard the supernatant solution.

    12. Rehydrate the pellet in High-Salt TE buffer.

    13. Add two volumes of 100% ethanol to the solution and mix gently.

    14. Centrifuge for 5 minutes at 4,000g. Discard the supernatant solution.

    15. Add (up to the original volume) cold 80% ethanol and centrifuge for 5 min. Discard the supernatant solution.

    16. Remove residual ethanol on the wall of microfuge tube by Kimwipe.
  • Air dry until all of the liquid has evaporated. Do not over-dry.

    17. Rehydrate in 100-500 ul of TE buffer.

    18.To remove RNA contamination, incubate the nucleic acids with 1/10th volume of RNase Stock Solution at 37oC for 1 hour.




    NOTES

  • The volume of 2 X Extraction Buffer added should be approximately equal to the volume of the tissue. If drier tissues are being used (e.g., grass leaves or seeds), one to two volumes of 1 X Extraction Buffer must also be added.


  • High molecular weight DNA is not as soluble as lower molecular weight DNA or RNA.


  • Frozen powdered tissue can be stored at -70oC until needed. Alternatively, large amounts of tissue can be powdered in liquid nitrogen using a Waring blender with a stainless steel jar.


  • When extracting from larger amounts of tissue, it is often easier to recover the DNA/CTAB precipitate with a hooked glass rod than to pellet it.


  • DNA from grass leaves that appears undegraded when examined by agarose gel electrophoresis immediately after preparation or after storage at -20oC may sometimes appear highly degraded after storage at 4oC for several days. Residual nuclease is the apparent cause of this. It is therefore recommended that grass DNA be stored at -70oC.





  • KIT INFORMATION




    REFERENCES

  • Bennett MD, Smith JB, Heslop-Harrison JS (1982) Nuclear DNA amounts in angiosperms. Proc. R. Soc. Lond. B. 216: 179-199.

  • Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version 11. Plant Mol. Biol. Rep. 1: 19-21.

  • Jones MC, Boffey SA (1984) Deoxyribonuclease activities of wheat seedlings. FEBS Lett. 174: 215-218.

  • Murray HG, Thompson WF (1980) Rapid isolation of high molecular weight DNA. Nucleic Acids Res. 8: 4321-4325.

  • Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol. Biol. 5: 69-76.

  • Zimmer EA, Newton KJ (1982) A simple method for the isolation of high molecular weight DNA from individual maize seedlings and tissues. In: "Maize for Biological Research", Sheridan WF (ed), Grand Forks, ND: University Press, University of North Dakota.


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

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