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
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.