[1] isolation of difficult plant DNA 3

Subsection A
Accessing the Templates

[1] Isolation of DNA from Plants with Large Amounts

of Secondary Metabolites
By Elizabeth A. Friar

Abstract
Many plant species have high contents of polysaccharides, polyphenols,
or other secondary metabolites that can interfere with DNA extraction
and purification. These contaminating compounds can lead to poor DNA
yield and prevent access by modifying enzymes, such as restriction endo-
nucleases and Taq polymerase. A number of factors, including choice
of plant tissue, tissue preparation, and modifications of the extraction
buffer, can help in DNA extraction for difficult plant species. This chapter
presents some of the DNA extraction protocols developed for various
plants.

Introduction
The tissues of many plant species contain large amounts of second-
ary compounds. These compounds may be derived from various bio-
synthetic pathways and play many roles in the biology of the organism.
Secondary metabolites may play important roles in plant defense, as
chemotoxins to potential fungal, insect, or vertebrate predators. Other
plant secondary compounds, particularly complex polysaccharides, play
an important role in plant osmoregulation and protection from desicca-
tion. Consequently, complex polysaccharides can make up a high percent-
age of tissue wet weight in cacti and other plants adapted to very dry
environments.
The presence of these plant secondary compounds can make
DNA extraction problematic. Some of them will coprecipitate with DNA
during extraction and inhibit further enzymatic modification of the DNA,
including restriction endonuclease digestion and polymerase chain reac-
tion (PCR) (Guillemaut and Marechal-Drouard, 1992). Large amounts of
complex polysaccharides can make extraction of usable DNA impossible,
rendering the aqueous portion of the extraction too viscous to allow for

Copyright 2005, Elsevier Inc.

All rights reserved.
METHODS IN ENZYMOLOGY, VOL. 395 0076-6879/05 $35.00
4 comparing macromolecules [1]

efficient separation of DNA from the contaminating polysaccharides.

These polysaccharides can also tightly adhere to the DNA, preventing
access by modifying enzymes (de la Cruz et al., 1997).
A number of methods have been developed to dilute, selectively pre-
cipitate, or inactivate the contaminating substances. For removal of poly-
saccharides, higher concentrations of cetyltrimethylammonium bromide
(CTAB), either in the initial extraction step or in a stepwise fashion over
several steps, help to selectively precipitate DNA. Higher concentrations
of CTAB and the addition of polyvinylpyrrolidone (PVP) or polyvinylpo-
lypyrrolidone (PVPP) can help to remove polyphenols. Additionally, some
protocols suggest the use of ascorbic acid, diethyldithiocarbamic acid
(DIECA), and 2-mercaptoethanol to reduce oxidation and prevent DNA
degradation.
The relative success of different protocols appears to be largely taxon
dependent; what may work for one taxon may not work for another. Here,
I present a number of protocols optimized for several taxonomic groups. It
may be necessary to try several approaches to achieve high yields of clean
DNA from a given species.

Tissue Choice
The choice of tissue for DNA extraction can be critical. Typically, the
younger the tissue, the lower the amounts of secondary compounds. Thus,
very young leaf tissue or newly germinated seedlings may be good choices
for DNA extraction. Additionally, flower buds, petals, epidermal layer, or
other soft tissues may be superior, particularly for plants such as cacti in
which stem tissue is highly mucilaginous. However, at least one protocol
has been developed to extract DNA from bark or young wood of woody
species, allowing for identification of cultivars or rootstocks in winter
condition (Cheng et al., 1997; see below for protocol).

Tissue Preparation
Typically, the more finely ground the tissue before DNA extraction, the
better the extraction of DNA from the contaminating compounds. This can
be accomplished by fine grinding in a mortar and pestle of tissues frozen in
liquid nitrogen. Highly mucilaginous tissues, however, may become too
hard to grind by hand. In those cases, it is possible to coarse chop frozen
tissues in a blender or food processor, followed by hand grinding. Lyophi-
lization of plant tissues can also aid in DNA extraction and minimize the
amount of contaminating substances. In this case, the plant tissue can be
[1] isolation of difficult plant DNA 5

lyophilized and stored, then ground to a fine powder in a bead mill, and
extracted using a standard CTAB extraction protocol.

DNA Yield
Yield of DNA from several of these extraction methods can be
quite low. Barnwell et al. (1998) found that using the same protocol, pea
shoots yielded 300 g of DNA per gram of fresh tissue, but Sedum tele-
phium yielded only 20 g/g of fresh tissue. Some of this difference can be
attributed to the high water content of many succulent plants. In addition,
some DNA will be inevitably lost because it has been complexed with
polysaccharides or polyphenols and lost during the extraction procedure.
The more purification steps requiring differential precipitation or extrac-
tion, the more DNA will be inevitably lost. Thus, protocols that can
successfully remove contaminants from genomic DNA in as few steps as
possible will provide the best yields.
It should also be noted that samples yielding low amounts of DNA
should be redissolved in distilled water rather than TE buffer, if PCR
applications will be used downstream. If yields of DNA are low, the normal
practice of dissolving the pellet in TE for a stable stock solution and
diluting an aliquot in water for PCR may lead to levels of ethylenediami-
netetraacetic acid (EDTA) in the PCR stock sufficient to inhibit Taq
polymerase activity.

RNA Removal
All of the protocols presented here will coprecipitate DNA and
RNA. Retention of contaminating RNA in DNA extractions can inflate
measures of DNA concentration, because of the overlapping fluores-
cence peaks. RNA may also complicate downstream PCR protocols by
selectively amplifying shorter complementary DNA (cDNA) copies rather
than the desired genomic copies of genes. RNA contamination may be
particularly problematic in random amplified polymorphic DNA (RAPD)
protocols.
RNA can be removed by incubation with RNase A, either after the
nucleic acids have been extracted or by inclusion of RNase A in the initial
extraction buffer. RNA may also be removed by differential precipi-
tation using lithium chloride. This protocol will remove larger RNA
fragments, and smaller RNA species (e.g., tRNAs) may remain in the
solution with the genomic DNA. (See detailed protocols in the following
sections.)
6 comparing macromolecules [1]

Methods: Isolation of DNA

Method 1: Isolation of DNA from Mucilaginous Tissues (Large
Preparation)
1. Preheat 2 CTAB buffer (2% CTAB, 1.4 M NaCl, 0.2% 2-
mercaptoethanol, 20 mM EDTA, 0.1 M Tris–HCl, pH 8.0) to 60 . Aliquot
25 ml of preheated buffer into a 50-ml conical tube for each DNA
extraction.
2. Grind about 5 g of fresh tissue to a fine powder using one of the
methods described earlier in the section ‘‘Tissue Preparation.’’
3. Immediately brush ground powder into the preheated isolation
buffer and place in a 60 water bath.
4. Incubate at 60 for 30 min.
5. Add an equal volume of chloroform:isoamyl alcohol (24:1) to each
tube and agitate for 10 min. Adequate mixing of both layers is vital at this
stage.
6. Centrifuge tubes at 6000 rpm for 5 min.
7. Transfer supernatant to a new 50-ml tube, being careful not to
disturb the white layer between phases.
8. Add 2/3 volume of ice-cold isopropanol to supernatant and gently
rock tube to precipitate DNA.
9. Collect DNA by pelleting or spooling. Pellet DNA by centrifu-
gation for 2 min at 4000g. Spool by collecting long strands of DNA using a
glass rod or pipette with a bent tip (a DNA hook can be made by gently
heating the narrow end of a disposable glass pipette and bending it into a
hook shape). Spooling selectively extracts high-molecular-weight DNA
and reduces the amount of coprecipitating contaminants.
10. Transfer pellet to one or two 1.5-ml Eppendorf tubes filled with
0.75 ml of 60 2 CTAB buffer. Use of two tubes allows for better
agitation and increases yield. Mix well.
11. Incubate tubes at 60 for 30 min, mixing occasionally.
12. Add an equal volume of chloroform:isoamyl alcohol (24:1) and
agitate well for 10 min.
13. Centrifuge in a Microfuge at maximum speed for 5 min.
14. Transfer supernatant to a new 1.5-ml tube.
15. Add 2/3 volume of ice-cold isopropanol and gently rock tubes to
precipitate DNA.
16. Collect DNA by centrifugation in a microcentrifuge at maximum
speed for 3 min.
17. Carefully pour off isopropanol, rinse with 70% ethanol, and
centrifuge at maximum speed for 2 min.
[1] isolation of difficult plant DNA 7

18. Pour off ethanol and dry pellet thoroughly in a vacuum centrifuge
or vacuum oven.
19. Resuspend pellet in 200 l of TE or diH2O.
Method 2: Isolation of DNA Using Starch Digestion
1. Mix 2 CTAB (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 0.1 M
Tris–HCl, pH 8.0) buffer with 1% w/v caylase (Cayla, Inc.). Just before
using, add 1% 2-mercaptoethanol.
2. Cut approximately 1 g of leaf tissue into small pieces and then
grind in liquid nitrogen. Add 7 ml of the CTAB mixture to the powder and
mix to form a slurry. Pour the slurry into a 15-ml polypropylene tube.
3. Cap the tube loosely and incubate at 65 for 30–60 min without
shaking.
4. Add 5 ml of chloroform:isoamyl alcohol (24:1) to each tube, cap
tightly, and mix well.
5. Centrifuge tubes at 7000g for 10 min.
6. Using a wide-bore pipette, transfer the supernatant to a clean,
labeled 30-ml Corex tube. Add 5 ml of ice-cold isopropanol. Cap the tube
and swirl gently to precipitate the DNA. Place at 20 for at least 1 h.
7. Centrifuge the tubes at 7000g at 4 for 30 min to pellet the DNA.
Pour off the supernatant and dry pellets in a vacuum oven for 10 min.
8. Dissolve the pellet in 1.5 ml of distilled water. Place in a 37 water
bath for 10–30 min, mixing the tube occasionally to dissolve the pellet. The
pellet will probably not dissolve completely. Transfer pellet and solution to
a 15-ml polypropylene tube.
9. Add 7 ml of CTAB to each tube (without caylase or 2-
mercaptoethanol). Cap tubes loosely and incubate at 65 for 30 min.
10. Repeat steps 4, 5, and 6.
11. Centrifuge tubes at 7000g at 4 for 30 min. Add 5 ml of ice-cold
EtOH-NH4OAc (10 mM NH4OAc in 76% EtOH) to each tube to pellet
DNA. Swirl gently, then let stand at room temperature for 10 min. Pour
off supernatant and dry the pellet in a vacuum oven for 10 min. Be careful
not to pour off the pellet, because it may be loose.
12. Resuspend pellet in 0.2 ml of diH2O or TE at 37 and transfer
solution to a 1.5-ml Microfuge tube.
Method 3: DNA Isolation Using Nucleon PhytoPure Resin
(Micropreparation)
1. Grind 0.1 g of frozen plant tissue in liquid nitrogen to form a fine
powder.
2. Transfer powder to a 1.5-ml Microfuge tube.
8 comparing macromolecules [1]

3. Add 500 l of CTAB extraction buffer 1 to each tube (500 l

2 CTAB [0.1 M Tris–HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, pH 8.0,
2% w/v CTAB, 1% w/v PVP-40, 1% w/v sodium bisulfite, 0.2% 2-
mercaptoethanol], 2 l of 20 mg/ml proteinase K, 2 l 2-mercaptoetha-
nol). Mix well by vortexing or by inversion.
4. Incubate tubes at 50 for 20–30 min, mixing every 5 min.
5. Transfer tubes to 65 and incubate another 15 min.
6. Remove tubes from water bath and incubate on ice for 2–3 min.
7. Add 500 l of ice-cold 100% chloroform to each tube, then add
100 l nucleon resin, using a wide-bore pipette tip. Invert tubes to mix,
degas once, and then close lids tightly.
8. Gently shake or rock tubes for 15 min at room temperature.
9. Centrifuge tubes in a microcentrifuge at maximum speed for 10 min.
10. Transfer supernatant to a 2-ml tube. (Do not use a 1.5-ml tube, as
the additional volume is necessary for optimal mixing.)
11. Add 1 ml of ice-cold 95% ethanol to each tube to precipitate DNA.
Incubate tubes at 20 for at least 1 h or overnight.
12. Centrifuge tubes in a microcentrifuge at maximum speed for
10 min to pellet DNA.
13. Pour off supernatant.
14. Dry pellets in a vacuum centrifuge for 10–20 min.
15. Resuspend pellets in 150 l of diH2O and incubate at 37 for
10–20 min, mixing every 5 min.
16. Add 500 l of CTAB extraction buffer 2 to each tube (500 l
2 CTAB, 1% w/v caylase) and invert to mix. Incubate tubes at 65 for
30 min.
17. Remove tubes from water bath and place on ice for 3–5 min.
18. Add 500 l of ice-cold 100% chloroform to each tube. Invert tubes
to mix, degas once, and then close lids tightly.
19. Centrifuge tubes in a microcentrifuge at maximum speed for
10 min.
20. Transfer supernatant to new 2-ml tube.
21. Add 1 ml of ice-cold 95% ethanol to each tube, invert gently to
precipitate DNA. Incubate tubes at 20 for at least 1 h. An overnight
incubation is preferable.
22. Centrifuge tubes in a microcentrifuge at maximum speed for
10 min. Discard supernatant.
23. Wash pellet by adding 500 l of 75% ethanol to each tube and
rocking gently for 10 min.
24. Centrifuge tubes in a microcentrifuge at maximum speed for 5 min.
Discard supernatant.
25. Dry pellet in a vacuum centrifuge for 10–20 min.
[1] isolation of difficult plant DNA 9

26. Resuspend pellets in 100 l of TE or diH2O and incubate at 37 for

10–20 min, mixing every 5 min.
Method 4: Fragaria DNA Extraction (Porebski et al., 1997)
1. Grind 0.5 g of frozen leaf tissue in liquid nitrogen until finely
ground. Transfer powder to 15-ml polypropylene tubes.
2. Add 5 ml of 60 extraction buffer (100 mM Tris–HCl, pH 8.0,
1.4 M NaCl, 20 mM EDTA, pH 8.0, 2% w/v CTAB, 0.3% 2-mercap-
toethanol) and 50 mg of PVP-40 to each tube. Mix by inversion and
incubate at 60 with shaking for 25–60 min.
3. Remove tubes from oven and allow to cool to room temperature.
4. Add 6 ml of chloroform:octanol (24:1) to each tube and mix well.
5. Centrifuge tubes at 3000 rpm for 20 min.
6. Transfer aqueous phase to a new polypropylene tube and repeat
chloroform:octanol extraction once more to remove cloudiness in the
aqueous phase caused by the PVP. Repeat steps 4, 5, and 6 until the
aqueous phase is no longer cloudy.
7. Add 1/2 volume of 5 M NaCl to final aqueous solution. Mix well.
Add 2 volumes of ice-cold 95% ethanol to precipitate DNA. If required,
incubate tubes at 20 for 20 min to overnight to aid precipitation.
8. Centrifuge tubes at 3000 rpm for 6 min to pellet DNA.
9. Pour off supernatant and wash pellet with ice-cold 70% ethanol.
Dry pellet in 37 oven or vacuum oven.
10. Dissolve pellets in 300 l of TE overnight at 4 to 6 . Transfer
DNA to 1.5-ml Eppendorf tubes. (Note: DNA is usually not amplifiable
at this stage.)
11. Add 3 l of RNase A (10 mg/ml) to each tube and incubate at 37
for approximately 1 h. Add 3 l proteinase K (1 mg/ml), and incubate for
an additional 15–30 min.
12. Add 300 l of phenol:chloroform (1:1) to each tube. Vortex well
to mix.
13. Centrifuge tubes in microcentrifuge at maximum speed for
10–20 min.
14. Remove top layer to a new 1.5-ml tube. Add 50 l of TE to the
phenol phase and recentrifuge as above. Collect top layer and add to the
previously collected top layer.
15. Add 1/10 volume 2 M NaOAc and 2 volumes absolute ethanol to
sample and mix well. Incubate tubes overnight at 20 .
16. Centrifuge tubes in a microcentrifuge at maximum speed for
10–20 min. Pour off supernatant and wash pellet with 70% ethanol. Dry
pellet in 37 oven or vacuum oven.
10 comparing macromolecules [1]

17. Add 100–200 l of TE or diH2O to each tube. Incubate overnight

at 4 for complete resuspension.
Method 5: Cactus DNA Extraction (de la Cruz et al., 1997)
1. Grind 3 g of tissue to a fine powder in liquid nitrogen.
2. Add 4 ml of CTAB extraction buffer (100 mM Tris–HCl, pH 8.0,
20 mM EDTA, pH 8.0, 4% CTAB, 1.5 M NaCl, 4% PVP-40, 500 g
ascorbic acid, 500 g DIECA, 10 mM 2-mercaptoethanol) with further
grinding to produce a slurry.
3. Add 15 ml of STE extraction buffer (100 mM Tris–HCl, pH 8.0,
50 mM EDTA, pH 8.0, 100 mM NaCl, 10 mM 2-mercaptoethanol) and
transfer the solution to a 50-ml tube.
4. Add 1 ml of 20% sodium dodecyl sulfate (SDS) and shake
vigorously for 7 min.
5. Incubate at 65 for 10 min.
6. Add 5 ml of cold 5 M potassium acetate and incubate at 0 for
40 min.
7. Centrifuge tubes at 20,000 rpm for 20 min to remove debris.
8. Filter the aqueous phase through a Miracloth filter into a clean
50-ml tube.
9. Add 7/10 volume of ice-cold isopropanol, mix gently, and incubate
at 20 for 10 min to precipitate genomic DNA.
10. Centrifuge tubes at 20,000 rpm for 15 min and discard the super-
natant.
11. Air-dry the pellet and resuspend in 1 ml of TE.
12. Transfer the solution to a 1.5-ml tube and centrifuge in a microcen-
trifuge at full speed for 10 min.
13. Transfer the supernatant to a new 1.5-ml tube and add 65 l of 3 M
sodium acetate and 600 l of ice-cold isopropanol and gently mix.
14. Incubate at 20 for 10 min.
15. Centrifuge tube in a microcentrifuge at full speed for 30 s.
16. Wash the pellet carefully with 76% ethanol.
17. Resuspend pellet in 1 ml of TE or diH2O.
Method 6: Cactus DNA Extraction 2, from Roots (Tel-Zur et al., 1999)
1. Rinse 0.5–1.0 g of root material with distilled water.
2. Grind the tissue to a fine powder in a mortar and pestle under liquid
nitrogen. Transfer the powder to a 50-ml tube.
3. Add 20 ml of extraction buffer to each tube (100 mM Tris–HCl,
pH 8.0, 0.35 M sorbitol, 5 mM EDTA, pH 8.0, 1% 2-mercaptoethanol),
keeping tubes on ice.
[1] isolation of difficult plant DNA 11

4. Centrifuge tubes at 10,000g for 10 min at 4 .

5. Pour off the supernatant carefully. Redissolve the pellet with 20 ml
of extraction buffer and recentrifuge. Repeat once more.
6. Pour off the supernatant again and redissolve the pellet in 5 ml of
extraction buffer. Add 3.5 ml of high-salt CTAB buffer (50 mM Tris–HCl,
pH 8.0, 4 M NaCl, 1.8% CTAB, 25 mM EDTA, pH 8.0) and 0.3 ml of
Sarkosyl 30% to each tube. Incubate mixture at 55 for 60–90 min.
7. Add equal volume of chloroform:isoamyl alcohol and mix well.
Centrifuge tubes at 10,000g for 10 min. Transfer supernatant to a Corex
tube.
8. Add 2/3 volume ice-cold absolute isopropanol and then 1/10
volume 3 M sodium acetate, pH 5.2.
9. Pour off supernatant and wash the pellet with ice-cold 75%
ethanol.
10. Air-dry the pellets and dissolve in 200 l of TE or diH2O.
11. Perform RNase digestion as below.

Method 7: Sedum DNA Extraction (Barnwell et al., 1998)

1. Grind leaves to a fine powder in a mortar and pestle under liquid
nitrogen. Transfer powder to a 50-ml tube.
2. Add 5 volumes of extraction buffer (100 mM Tris–HCl, pH 8.0,
20 mM Na2EDTA, 2% w/v CTAB, 1.4 M NaCl, 1% w/v PVP-40) and mix
to form a paste.
3. Incubate mixture at 65 with occasional shaking for 10 min.
4. Centrifuge tubes at 3000g for 5 min.
5. Transfer the supernatant to a new tube and add 1.25 times the
original tissue volume of 10% w/v CTAB (in 0.7 M NaCl). Vortex the
mixture well.
6. Centrifuge tubes at 3000g for 5 min.
7. Transfer supernatant to a new tube and add 3 volumes of
precipitation buffer (50 mM Tris–HCl, pH 8.0, 1 mM Na2EDTA, 1.0 M
NaCl) and mix well.
8. Incubate tubes at room temperature (22 ) for 30 min.
9. Centrifuge tubes at 5000g for 15 min. Discard the supernatant.
10. Dissolve the pellet in high-salt TE buffer (10 mM Tris–HCl, pH
8.0, 1 mM Na2EDTA, 1 M NaCl). Transfer to a microcentrifuge tube.
11. Add 2 volumes of ice-cold 95% ethanol to precipitate nucleic acids.
Incubate at 20 for 1 h.
12. Centrifuge tubes in a microcentrifuge at full speed for 5 min to
pellet nucleic acids.
13. Wash the pellet twice with 70% ethanol.
14. Dissolve pellet in 100 l of diH2O.
12 comparing macromolecules [1]

Method 8: DNA Extraction from Woody Material (Bark, Dormant

Buds, Young Stems; Cheng et al., 1997)
1. Grind 0.05–0.1 g of material to a fine powder with liquid nitrogen.
2. Add 1 ml of extraction buffer (100 mM Tris–HCl, pH 8.0, 20 mM
EDTA, pH 8.0, 1.5 M NaCl, 2% w/v CTAB, 2% w/v PVP-40T) to the
powder and transfer solution to a 1.5-ml tube.
3. Incubate tubes at 65 for 30 min.
4. Add enough chloroform:isoamyl alcohol (24:1) to almost fill the
tube. Shake vigorously to form an emulsion.
5. Centrifuge tubes at 8000g for 5 min.
6. Transfer aqueous phase to a new 1.5-ml tube and add 1 ml of
ice-cold 95% ethanol.
7. Incubate tubes at 20 for 5 min to precipitate DNA.
8. Centrifuge tubes at 8000g for 5 min. Pour off supernatant.
9. Add 600 l 1 M NaCl and incubate at 65 for 10 min to dissolve
DNA.
10. Add 300 l of phenol and mix gently.
11. Centrifuge tubes at 8000g for 3 min.
12. Transfer supernatant to a new 1.5-ml tube and add 500 l of chloro-
form to each tube.
13. Centrifuge tubes at 8000g for 5 min.
14. Transfer supernatant to a new 1.5-ml tube and add 1 ml of ice-cold
95% ethanol.
15. Incubate at 20 for 30 min.
16. Centrifuge tubes at 8000g for 10 min. Pour off supernatant.
17. Dissolve pellet in 600 l of diH2O and add 6 l of 0.1 M spermine.
Incubate on ice for 20 min.
18. Centrifuge tubes at 8000g for 10 min. Pour off supernatant.
19. Add 500 l 1 spermine pellet extraction buffer (10: 3 M sodium
acetate, 0.1 M magnesium acetate), diluted in 75% ethanol. Incubate tubes
on ice for 1 h.
20. Drain off liquid and wash pellet with 500 l of 75% ethanol.
21. Dry pellet either by air-drying or in a vacuum centrifuge.
22. Redissolve pellet in TE or diH2O.

Method 9: Isolation and Purification of DNA Using Sephacryl (Li

et al., 1994)
1. Grind 0.2 g of fresh tissue to a fine powder under liquid nitrogen.
2. Transfer powder to a 13-ml Röhrer tube containing 6 ml of extrac-
tion buffer (100 mM Tris–HCl, pH 8.0, 20 mM EDTA, 500 mM NaCl)
on ice.
[1] isolation of difficult plant DNA 13

3. Add 10 l of 2-mercaptoethanol and 400 l of 20% SDS. Mix

gently.
4. Incubate tubes at 65 for 10 min.
5. Add 2 ml of 3 M potassium acetate buffer, pH 5.2. Mix gently and
incubate on ice for 10 min.
6. Centrifuge tubes at 10,000g for 10 min.
7. Pour supernatant through 2 layers of Miracloth into a clean Röhrer
tube. Add 4 ml of isopropanol and mix gently. Incubate at 20 for
30 min.
8. Centrifuge tubes at 10,000g for 10 min to pellet DNA. Pour off the
supernatant and dry by inverting the tube over paper towels.
9. Dissolve pellet in 200 l buffer and transfer solution to a 1.5-ml tube.
10. Add 1 l RNase A (10 mg/ml) and incubate tubes at 65 for
10 min.
11. Put a small amount of glasswool in the bottom of a 1-ml plastic
syringe.
12. Fill the syringe to 0.7 ml with Sephacryl S-1000.
13. Place the syringe in a 13-ml Röhrer tube and centrifuge at
150–300g for 1 min.
14. Run approximately 300 l of STE buffer (100 mM NaCl, 10 mM
Tris–HCl, pH 8.0, 1 mM EDTA, 20% w/v PEG 8000 in 1.2 M NaCl)
through the syringe twice, centrifuging at 150–300g for 2 min each time.
15. Place the prepared spin column into a 13-ml Röhrer tube, making
sure the tip of the syringe is in the Eppendorf tube.
16. Load the DNA solution into the column and centrifuge at 150–300g
for 2 min.
17. Add 200 l of STE to the column and recentrifuge as above.
18. Add 200 ml of 20% PEG 8000 in 1.2 M NaCl to the DNA solution
and incubate on ice for 20 min.
19. Centrifuge tubes at 10,000g for 15 min at 4 .
20. Wash the pellets once using 70% ethanol.
21. Dissolve pellet in 100–500 l of TE or diH2O.

Methods: Removal of RNAs from Genomic DNA

Method 1: Differential Precipitation with Lithium Chloride
1. Add 0.2 volumes of ice-cold 12 M lithium chloride (LiCl) to
resuspended DNA.
2. Incubate at 4 for 30 min.
3. Centrifuge tubes for 10 min at 4 .
4. Discard pellet and retain supernatant.
14 comparing macromolecules [1]

Method 2: Digestion with RNase A (Tel-Zur et al., 1999)

1. Add 10 l of 5 mg/ml RNase A to 200 l of resuspended DNA
in TE.
2. Incubate tubes for 40 min at 37 .
3. Transfer solution to a 1.5-ml Eppendorf tube and add an equal
volume of phenol:chloroform (1:1 v/v).
4. Centrifuge tubes at maximum speed in a microcentrifuge for 10 min.
5. Transfer aqueous phase to a new 1.5-ml tube and add an equal
volume of cold chloroform.
6. Centrifuge tubes at maximum speed in a microcentrifuge for 10 min.
7. Remove the aqueous phase and precipitate the DNA with
2 volumes of ice-cold absolute ethanol mixed with 1/10 volume of
3 M sodium acetate, pH 5.2.
8. Incubate tubes at 20 for 30 min.
9. Centrifuge tubes at maximum speed in a microcentrifuge for 15 min.
10. Rinse the pellet with ice-cold 75% ethanol and allow pellets to
air-dry.
11. Dissolve pellet in 50–100 l of TE or diH2O.

References
Barnwell, P., Blanchard, A. N., Bryant, J. A., Smirnoff, N., and Weir, A. F. (1998). Isolation of
DNA from the highly mucilaginous succulent plant Sedum telephium. Plant. Mol. Biol.
Reptr. 16, 133–138.
Cheng, F. S., Brown, S. K., and Weeden, N. F. (1997). A DNA extraction protocol from
various tissues in woody species. HortScience 32, 921–922.
de la Cruz, M., Ramirez, F., and Hernandez, H. (1997). DNA isolation and amplification from
cacti. Plant Mol. Biol. Reptr. 15, 319–325.
Guillemaut, P., and Marechal-Drouard, L. (1992). Isolation of plant DNA: A fast, inexpensive
and reliable method. Plant Mol. Biol. Reptr. 10, 60–65.
Li, Q.-B., Cai, Q., and Guy, L. (1994). A DNA extraction method for RAPD analysis from
plants rich in soluble polysaccharides. Plant Mol. Biol. Reptr. 12, 215–220.
Porebski, S., Bailey, L. G., and Baum, B. R. (1997). Modification of a CTAB DNA extraction
protocol for plants containing high polysaccharide and polyphenol components. Plant Mol.
Biol. Reptr. 15, 8–15.
Tel-Zur, N., Abbo, S., Myslabodski, D., and Mizrahi, Y. (1999). Modified CTAB procedure
for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus
(Cactaceae). Plant Mol. Biol. Reptr. 17, 249–254.