Molecular Techniques and Methods

Isolation of Chloroplasts and Chloroplast DNA

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

In all land plants and algae, chloroplast DNA (cpDNA) has been found to exist as a single circular of molecules ranging from 80 to 300 kb. The chloroplast genome is densely packed with genes. Chloroplast gene products identified function either in photosynthesis or as components of the chloroplast protein synthesizing system.
In this procedure, sucrose step gradient procedure is used to isolate chloroplast and cpDNA.




MATERIALS AND SOLUTIONS

Homogenization Buffer (1 liter)
0.35 M Sorbitol ---------------------------------- 63.8 g
50 mM Tris-HCl (pH 8.0) ----------------------- 50 ml of 1 M Tris-HCl
5 mM EDTA ------------------------------------ 10 ml of 0.5 M EDTA
0.1% BSA --------------------------------------- 1 g
Deionized H2O to make a final volume of ------- 1 liter
  • Add 1 ml 2-mercaptoethanol just prior to use.


    Rinse Buffer (100 ml)
    0.35 M Sorbitol ----------------------------------- 6.38 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    25 mM EDTA ------------------------------------ 5 ml of 0.5 M EDTA
    Deionized H2O to make a final volume of -------- 100 ml


    Prechilled 1 liter Waring Blender


    52% Sucrose Gradient Buffer (100 ml)
    Sucrose ----------------------------------------- 52 g
    50 mM Tris-HCl (pH 8.0) ----------------------- 5 ml of 1 M Tris-HCl
    25 mM EDTA ----------------------------------- 5 ml of 0.5 M EDTA
    Deionized H2O to make a final volume of ------ 100 ml


    30% Sucrose Gradient Buffer (100 ml)
    Sucrose ---------------------------------------- 30 g
    50 mM Tris-HCl (pH 8.0) ---------------------- 5 ml of 1 M Tris-HCl
    25 mM EDTA ---------------------------------- 5 ml of 0.5 M EDTA
    Deionized H2O to make a final volume of ------ 100 ml



    Lysis Buffer (100 ml)
    5% Sodium sarcosinate (w/v) --------------------- 50 ml of 10% Sodium sarcosinate
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    25 mM EDTA ------------------------------------ 5 ml of 0.5 M EDTA
    Deionized H2O ----------------------------------- 40 ml


    10 X Ethidium Bromide Buffer (10 ml)
    Ethidium bromide -------------------------------10 mg
    500 mM Tris-HCl (pH 8.0) --------------------- 5 ml of 1 M Tris-HCl
    250 mM EDTA --------------------------------- 5 ml of 0.5 M EDTA


    NaCl-saturated Isopropanol
    Saturate isopropanol with 10 mM NaCl and H20.


    Dialysis Buffer (2 liter)
    10 mM Tris-HCl (pH 8.0) ----------------------- 20 ml of 1 M Tris-HCl
    10 mM NaCl ------------------------------------ 4 ml of 5 M NaCl
    0.1 mM EDTA ---------------------------------- 0.4 ml of 0.5 M EDTA
    Deionized H2O ---------------------------------- 1,976 ml
  • Keep in 4oC.




    PROCEDURES

    1. Keep plants in the dark for 3-4 days to reduce chloroplast starch levels.

    2. Collect
    young, healthy, de-starched (critical for intact chloroplast isolation) leaves

    3. Wash leaves in tap water if visibly dirty and cut into small pieces by scissors.

    4. Place 100 g of cut leaves in 400 ml of ice-cold Homogenization Buffer.

    5. Homogenize in a prechilled 1 liter Waring blender for 5 second bursts, three times at high speed.

    6. Filter through four layers of cheesecloth and one layer of miracloth.

    7. Centrifuge at 1,000g for 15 minutes at 4oC.

    8. Resuspend the pellet in 40 ml Rinse Buffer by vigorous swirling of the centrifuge bottle.

    9. Make a step gradient consisting of 18 ml of 52% Sucrose Gradient Buffer overlayered with 7 ml of 30% Sucrose Gradient Buffer 1-2 days in advance.
  • If the step gradients are made on the day of extraction, the 30% sucrose overlay should be mixed rather vigorously with the 52% underlay in order to achieve a diffuse interface. This prevents overly tight packing of chloroplasts at the interface during centrifugation and reduces nuclear DNA contamination of the chloroplast band.


  • 10. Load 20 ml of the resuspended pellet onto a step gradient.

    11. Centrifuge the step gradients at 25,000 rpm for 1 hour at 4oC.

    12. Remove the chloroplast band at the 30-52% interface with a wide bore pipette.

    13. Dilute the chloroplast with 5-10 volumes Rinse Buffer.

    14. Centrifuge at 1,500 g for 15 minutes at 4oC to pellet the chloroplasts.

    15. Repeat steps 13 and 14.

    16. Resuspend the chloroplast pellet in Rinse Buffer to a final volume of 15 ml.

    17. Add 1/20th volume of pronase (20mg/ml, 2 hour self-digested at 37oC).

    18. After 2 minutes at room temperature, add 1/5th volume of Lysis Buffer.

    19. Mix by inverting the tube several times over a period of 15 minutes.
  • If necessary, the lysed chloroplasts can be stored at 4oC for several hours before proceeding with the next step.


  • 20. Centrifuge for 10 minutes at 2,000g, 4oC to remove residual starch and cell wall debris from the chloroplast lysate.

    21. Add 24 g of CsCl and 1/10th volume of 10 X Ethidium Bromide Buffer.

    22. Centrifuge for 14-16 hours at 45,000 rpm.

    23. Remove the DNA band from a centrifuge tube by using a syringe needle (gauge-21).

    24. Remove ethidium bromide by three extractions with NaCl-saturated Isopropanol.

    25. Remove the DNA-containing CsCl phase from below the isopropanol phase.

    26. Dialyze against at least three changes of 2 liters of Dialysis Buffer over a period of 1-2 days at 4oC.

    27. Remove any visible particulate matter by centrifuging at 8,500 rpm for 5 minutes in a microcentrifuge at room temperature.

    28. Store DNA at 4oC for short-term use.
  • Store DNA at -80oC for long-term use.




    NOTES

  • The physiological state of the starting leaf material is absolutely crucial to the success of a cpDNA extraction. Wherever possible, only the freshest, youngest, and healthiest green leaves should be used. It is far better to replant and wait for new growth than to extract from leaves that are beginning to yellow and senesce or that have been stressed during growth, e.g., that have wilted one or more times.


  • Some plants grown under high light intensities accumulate within their chloroplasts very high levels of starch that are very difficult to deplete even by prolonged dark treatment and that result in highly damaged chloroplasts and low cpDNA yields. In these cases the best solution is to grow the plants under moderate or low light intensities, e.g., under a greenhouse bench rather than on top of it.


  • If getting intact chloroplasts is difficult, incorporates 10-25% (w/v) PEG 4000 in the Homogenization Buffer and all subsequent Rinse Buffers.


  • 0.1% (w/v) Polyvinylpyrrolidone (PVP) in the Homogenization Buffer can serve as an effective adsorbants for tannins and other secondary plant compounds.


  • Addition of 0.1% (w/v) antifoam to the Homogenization Buffer is useful with plant material that foams excessively during homogenization.


  • Keeping blender blades very sharp, or using a self-made cutting unit with multiple razor blades spaced along a central metal rod, will allow efficient cellular disruption in the absence of the high shear forces that tend to destroy organelle structure.


  • The amount of chloroplasts loaded per sucrose gradient can significantly affect the ultimate purity of the cpDNA. The less chloroplast material loaded, the higher the purity of the resulting cpDNA, primarily because less nuclear DNA will be trapped in gradients that are not overloaded and in which chloroplasts do not pack too tightly at the 30-52% sucrose interface.





  • KIT INFORMATION




    REFERENCES

  • Bohnert, H.J., Crouse, E.J., and Schmitt, J.M., 1982, In "Encyclopedia of Plant Physiology, New Series " (Parthier, B., and Boulter, D., eds.), Vol. 14B, p. 475. Springer-Veriag, Berlin and New York.


  • Bowman, C.M., and Dyer, T.A., 1982, Anal. Biochem. 122,108.


  • Grant, D.M., Ginham, N.W., and Boynton, J.E., 1980, P.N.A.S., 77, 6067.


  • Edelman, M., Hallick, R., and Chua, N.-H., eds., 1982, Methods in Chloroplast Molecular Biology". Elsevier, Amsterdam.


  • Jones, J.D., Hulme, A.C., and Wooltorton, L.S.C., 1965, Phytochemistry 4, 659.


  • Manning, J.E., Wolstenholme, D.R., Ryan, R.S., Hunter, J.A., and Richards, O.C., 1971, P.N.A.S. 68, 1169.


  • Murray, M.G., and Thompson, W.F., 1980, Nucleic Acids Res. 8, 4321.


  • Saghai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A., and Allard, R.W., 1984, P.N.A.S. 81, 8014.


  • Tewari, K.K., and Wildman, S.G., 1966, Science 153, 1269.


  • Whitfeld, P.R., and Bottomley, W., 1983, Annu. Rev. Plant Physiol. 34, 279.



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