Molecular Techniques and Methods

Isolation of Nuclei and Nuclear DNA from Plant Tissues

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The purification of nuclei from plant tissues contain the following features.
(1) a pretreatment of the tissue to enhance cell disruption, such as immersion in cold ether.
(2) homogenization in the presence of a membrane stabilizing agent(s), such as hexylene glycol or sucrose.
(3) filtration to remove whole cells and large debris.
(4) differential lysis of organelles with Triton X-100 in the presence of divalent cations (Mg2+ or Ca2+).
(5) purification of the nuclei by density gradient centrifugation.




MATERIALS AND SOLUTIONS

Extraction Buffer (100 ml)
1 M Hexylene Glycol -------------------------------------- 11.8 g
(or, 1 M Sucrose ------------------------------------------ 34.2 g)
10 mM Tris-HCl (pH 7.2) --------------------------------- 1 ml of 1 M Tris-HCl
10 mM MgCl2 --------------------------------------------- 1 ml of 1 M MgCl2
5 mM 2-Mercaptoethanol (add just before use) ------------ 35 ul of 14.3 M 2-Mercaptoethanol
Add distilled H2O to make a final volume of -------------- 100 ml


Gradient Buffer Stock-A (100 ml)
Extraction Buffer ------------------------------------------- 99 ml
0.5% Triton X-100 ---------------------------------------- 1 ml of 50% Triton X-100


Gradient Buffer Stock-B (100 ml)
90% Percoll ----------------------------------------------- 90 ml of 100% Percoll
0.5 M Hexylene Glycol ------------------------------------ 5.9 g
(or, 1 M Sucrose ------------------------------------------ 34.2 g)
10 mM Tris-HCl (pH 7.2) -------------------------------- 1 ml of 1 M Tris-HCl
10 mM MgCl2 -------------------------------------------- 1 ml of 1 M MgCl2
5 mM 2-Mercaptoethanol (add just before use) ----------- 35 ul of 14.3 M 2-Mercaptoethanol
0.5% Triton X-100 --------------------------------------- 1 ml of 50% Triton X-100


2 x Lysis Buffer (100 ml)
0.2 M Tris-HCl (pH 8.0) ---------------------------------- 20 ml of 1 M Tris-HCl
0.1 M EDTA (pH 8.0) ------------------------------------ 20 ml of 0.5 M EDTA
1 M NaCl ------------------------------------------------- 20 ml of 5 M NaCl
2% Sarkosyl ----------------------------------------------- 20 ml of 10% Sarkosyl
Distilled H2O ---------------------------------------------- 20 ml


Pronase Solution (10 ml)
Protease Type XIV (Sigma P-5147) ----------------------- 100 mg
20 mM Tris-HCl (pH 7.5) --------------------------------- 0.2 ml of 1 M Tris-HCl
Distilled H2O ---------------------------------------------- 9.7 ml
  • Self-digest at 37oC for 1 hour.
  • Store at -20oC in small aliquots.


    CsCl Solution (100 ml)
    4.45 M CsCl ---------------------------------------------- 75 g
    0.05 M Tris-HCl (pH 8.0) --------------------------------- 5 ml of 1 M Tris-HCl
    0.025 M EDTA -------------------------------------------- 5 ml of 0.5 M EDTA
    Ethidium Bromide ------------------------------------------ 20 mg
    Add distilled H2O to make a final volume of -------------- 100 ml




    PROCEDURES

  • Perform all steps on ice!

    1. Germinate seeds and grow for 1-2 weeks under continuous white fluorescent light.

    2. Place the plants in a dark cabinet for 24 hours prior to harvesting to deplete the starch.

    3. Harvest leaves into a vessel submerged in ice, and then weigh.

    4. In a fume hood, treat the leaves with ice cold anhydrous Ether (3-5 ml/g fresh weight) for 2-3 min.

    5. Rinse away residual ether with ice cold Extraction Buffer (use 3 ml/g fresh weight).

    6. Add fresh cold Extraction Buffer to the leaves (3-5 ml/g fresh weight).

    7. Homogenize leaves with a Polytron (PT 10-35; Brinkman) at a medium setting for 1-2 min.
  • It is important not to grind to the point where frothing occurs.
  • Disruption of fibrous materials may require somewhat longer grinding times or higher speeds.

    8. Filter the homogenate through four layers of cheesecloth directly onto a 300 um nylon mesh.

    9. Add Triton X-100 dropwise, while gently swirling the filtrate.
  • The final Triton X-100 concentration should be 0.5%.

    10. Filter the lysate sequentially through 100 um, 50 um, and 20 um nylon mesh.
  • Use a flat Teflon spatula to flow through the 20 um mesh.

    11. Layer the filtered lysate over an appropriate set of Percoll Step Gradients, prepared in round-bottomed polycarbonate centrifuge tubes. Set the Percoll Step Gradients as follows.

    Gradients
    Solutions
    60% Percoll Gradient Solution
    3.3 ml of Gradient Buffer Stock-A
    6.7 ml of Gradient Buffer Stock-B
    30% Percoll Gradient Solution
    6.6 ml of Gradient Buffer Stock-A
    3.4 ml of Gradient Buffer Stock-B


  • For 10 g of each starting material, use one gradient consisting as follow in round-bottomed polycarbonate centrifuge tube.
    60% Percoll ----------- 8 ml
    30% Percoll ----------- 5 ml

    12. Layer the 20-35 ml sample solution on top of the step gradient.

    13. Centrifuge the gradients for 30 min at 200g.

    14. Remove the upper portions of the step gradient by pipetting.

    15. Resuspend the pellet (containing nuclei and starch) in 10-20 ml Gradient Buffer Stock-A.
  • High quality nuclei generally resuspend quite easily.
  • Starch can be frequently left behind, since it is much more difficult to resuspend.

    16. Layer the resuspended material over a 10 ml cushion of 60% Percoll Gradient Solution.

    17. Centrifuge the gradients for 30 min at 200g.

    18. Resuspend each pellet in 10 ml Gradient Buffer Stock-A.
  • When the isolation of DNA is the ultimate aim, washing away residual Percoll does not necessary.
  • To obtain good lysis, it is important to thoroughly resuspend the nuclei, which is usually done by gently pipetting the organelles in a serological pipette.

    19. For lysis, transfer 10 ml aliquots of nuclei to 50 ml screw-cap centrifuge tubes.
  • Add 10 ml of 2 x Lysis Buffer to each tube and mix in by gentle inversion.
  • The solution should become quite viscous at this point. Viscosity may continue to increase for 10-20 min.
  • Indeed, increase in viscosity upon lysis is a good marker for the position of nuclei in Percoll gradient fractions.

    20. Add 1/20 volume Pronase Solution, mix gently and incubate at 37oC for 30-60 min.

    21. Add second aliquot of Pronase Solution, and incubate for an additional 30-60 min.
  • Proteinase K (1 mg/ml) can be substituted for Pronase.

    22. Add finely ground solid CsCl to the digest, using 1.0 g CsCl/ml of lysate.
  • Dissolve the CsCl by gentle inversion.

    23. Add 320 ul Ethidium Bromide (10 mg/ml) per 10 ml of DNA-CsCl mixture.
  • Final density should be about 1.55 g/ml.

    24. Transfer the mixture to a polyallomer ultracentrifuge tube.

    25. Centrifuge for 12 hours at 20oC at 42,000-58,000 rpm.

    26. Examine the gradients under long wavelength UV light.
  • The DNA is observed as a fluorescent band near the middle of the gradient.

    27. Collect the DNA band from above with a wide-bore serological pipette to minimize shearing.

    28. Transfer the DNA to a plastic screw-cap centrifuge tube.
  • Extract the Ethidium bromide with several changes of isopropanol equilibrated with NaCl-saturated water or water-saturated butanol.

    29. Remove CsCl by dialysis against 1,000-fold volumes of TE at room temperature.
  • During dialysis, change TE at least 4 times.

    30. Centrifuge the dialyzed DNA for 10 min at about 200g to remove particulates if necessary.

    31. Determine the concentration of DNA spectrophotometrically at 260 nm.
  • 1 A260 unit = 50 ug double-stranded DNA.

    32. If necessary, the DNA can be further concentrated by precipitation with addition of 1/10 volume 3 M Sodium acetate (pH 5.5) and 2.5 volume cold 95% Ethanol.




    NOTES

  • Hexylene glycol (2-methyl-2,4-pentandiol) stabilizes isolated mitotic chromosomes. The chromatin within nuclei prepared in hexylene glycol buffers is packaged into a typical nucleosomal structure as determined by sensitivity to exogenous micrococcal nuclease. Further, the sensitivity of transcriptionally active chromatin to exogenous DNase I is greater than inactive chromatin.

  • The tissue is treated with cold Ether, which apparently enhances cell disruption. Ether may exert its effect at least partly through the removal of cuticular waxes, since a substantial waxy residue remains after evaporation of ether used to treat leaves.

  • In the presence of divalent cations, Triton does not solubilize nuclear DNA. Although nuclei exposed to Triton still retain a native chromatin structure and RNA polymerase activity, some membrane damage does occur as a result. Earlier reports indicated that only the outer of two nuclear membranes was stripped away as a result of Triton.

  • Nuclei are separated from other cellular debris by Density Gradient Centrifugation in suspensions of Percoll, a colloidal silica coated ith polyvinylpyrolidone. Percoll gradients are an attractive alternative to more traditional approaches using sucrose. Due to the low viscosity of Percoll, fractionations do not require ultracentrifugation.

  • Position of Nuclei on Percoll Gradients
    It is advisable to monitor the position of nuclei by microscopy when working with a new species. Fluorescence microscopy with DNA-specific dyeS21 is particularly useful in this regard.

  • Once the nuclei have been purified, extraction of DNA is accomplished by Detergent lysis and thorough Protease digestion. Failure to completely digest proteins at this point is the most frequent reason for subsequent inability to digest the DNA with restriction enzymes. DNA is separated from RNA and any residual protein by repeated CsCl-Ethidium bromide gradient centrifugation.




    KIT INFORMATION




    REFERENCES

  • Dunham VL, Bryant JA (1983) In "Isolation of Membranes and Organelles from Plant Cells" (Hall JL, Moore AL eds.) p.237. Academic Press, New York.

  • Murray MG, Cuellar RE, Thompson WF (1978) Biochemistry 17, 5781.

  • Murray MG, Palmer JD, Cuellar RE, Thompson WF (1979) Biochemistry 18, 5259.

  • Saghai-Maroof MA, Sullivan KM, Jorgensen RA, Allard RA (1984) PNAS 81, 8014.

  • Casey J, Davidson N (1977) Nucleic Acids Res. 4, 1539.

  • Birnboim HC, Doly J (1979) Nucleic Acids Res. 7, 1513

  • Timmis JN, Scott NS (1983) Nature (London) 305, 65.


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