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INTRODUCTION
Plant molecular biology research and crop molecular breeding require analysis of ge-
nomic DNAs (gDNAs). For some applications such as restriction analysis, gene cloning,
PCR amplification, sequencing of large fragments, and construction of genomic libraries,
higher-quality and relatively larger-size gDNAs are required. However, for large-scale
genotyping analysis, the high throughput of the gDNA preparation method is the first
priority. So far, many plant gDNA preparation methods have been developed for various
applications.
Preparation
of Plant gDNA
Some plant cells contain high amounts of secondary metabolites, such as polyphe-
nols, tannins, or polysaccharides, which are difficult to remove. Thus, some modifica-
tions should be included during gDNA preparation. For example, polyvinyl pyrrolidone
(PVP) helps to remove polyphenols by forming hydrogen-bonding complexes (John,
1992). NaCl can be applied to remove the polysaccharides (Lodhi et al., 1994). After
CTAB lysis, anion-exchange chromatography can also help to improve the quality of the
gDNA extracted from oak, elm, pine, fir, poplar, maize, and rhododendron (Csaikl et al.,
1998).
We introduce the original large-scale (maxi) CTAB preparation method in Basic Protocol
1 of this article, which yields high-quality DNA. In Basic Protocol 2, we describe a
modified SDS and CTAB mini-extraction method. Basic Protocol 3 provides a method
for preparation of megabase-size nuclear gDNA. In Basic Protocol 4, we describe a low-
cost, very-high-throughput method for rapid preparation of gDNA and application of the
template DNAs to PCR reactions in a 96-well format. These protocols are summarized
in Table 1.
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Volume 1 Current Protocols in Plant Biology
Materials
Rice plant tissue
Liquid nitrogen
CTAB extraction buffer (see recipe)
24:1 (v/v) chloroform/isoamyl alcohol
10% (w/v) CTAB
CTAB precipitation buffer (see recipe)
High-salt TE buffer, pH 8.0 (see recipe)
10 mg/ml RNase A (store at 20C)
Isopropanol (or ethanol)
70% ethanol
TE buffer, pH 8.0 (no NaCl; see recipe)
Lambda DNA
Restriction enzyme (e.g., HindIII, EcoRI)
4. Recover supernatant into a 50-ml centrifuge tube. Add 0.1 vol of 10% CTAB and
mix thoroughly.
Preparation
Be careful not to transfer the impurities and debris. of Plant gDNA
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Current Protocols in Plant Biology Volume 1
5. Extract with 15 ml 24:1 chloroform/isoamyl alcohol, mix, centrifuge, and recover
supernatant as in step 3 and 4 above.
Usually two rounds of chloroform extraction are sufficient for removing most impurities.
Additional chloroform extraction can be performed if the supernatant is still turbid.
Precipitation of DNA
6. Add 1 to 1.2 vol (with respect to volume of the recovered supernatant) precipitation
buffer and mix well. Incubate at room temperature for 10 to 15 min.
If precipitate (from >2 g starting material) is not visible, the salt concentration may
be too high; thus, add additional precipitation buffer up to 1.3 vol with respect to the
recovered supernatant.
7. Centrifuge 10 min at 6,000 g (4,000 rpm in Eppendorf F-34-6-38 rotor), room
temperature.
Do not increase the speed or time of centrifugation; otherwise, DNA pellets (white or
gray) may become difficult to dissolve in the following step.
8. Discard the supernatant and remove any residual liquid with a pipet. Add 4 to 5 ml
(1 ml per g of starting material) high-salt TE buffer and 5 l of 10 mg/ml RNase A.
Incubate sample at 55C with gentle shaking until the pellets dissolve completely.
9. Precipitate the DNA by adding 0.6 vol isopropanol (or 2 vol ethanol) and mixing
well. If flocculent DNA is visible, lift out the precipitated DNA with a pipet tip or
glass hook, and wash twice with 70% ethanol.
If DNA is prepared from less (<2 g) starting material, flocculent DNA may not appear.
In such cases, centrifuge the precipitation 5 to 10 min at 12,000 to 15,000 g to pellet
the DNA.
10. Air-dry the DNA (or DNA pellet) and dissolve the DNA in 0.3 to 0.5 ml TE buffer
(no NaCl) at 50 to 60C with gentle shaking.
Overly dried DNA is difficult to dissolve in TE buffer.
DNA dissolved in deionized water is unstable and not suitable for long-term storage.
Checking DNA concentration and quality
11. Measure the gDNA concentration using a spectrophotometer such as
NanoDrop2000.
Due to contamination of residual RNA, the DNA concentration value measured with a
spectrophotometer is often higher than the actual concentration.
12. Dilute a small part of the gDNAs to 100 ng/l based on the measurement above.
Take 2 l (estimated 200 ng) for agarose gel electrophoresis (Voytas, 2000) using
a 0.75% agarose gel with different amounts (e.g., 50, 100, 150, and 200 ng) of
lambda DNA as standards. Compare the bands of the gDNAs with the lambda DNA
standards to check the DNA quality and re-estimate the DNA concentrations.
High-quality DNA should show a major band that migrates similarly to the lambda DNA,
with less smear below the major band.
13. Optional: Prepare several test digestion reactions (20 l), each containing 1 g
gDNA and 5 U (no excess enzyme is added) of a restriction enzyme (such as
HindIII, EcoRI). After digesting at 37C for 3 hr, run the DNA on a 0.75% agarose
gel (Voytas, 2000).
An efficient restriction digest will give rise to a smeared DNA pattern in the gel, which
indicates that the quality of DNA is high.
Preparation
of Plant gDNA 14. Store the DNA at 20C.
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Volume 1 Current Protocols in Plant Biology
MINI PREPARATION OF PLANT GDNA BASIC
PROTOCOL 2
This protocol introduces a simple method to isolate rice plant total gDNA on a small
scale. A small amount (0.5 g) of plant tissue is ground with one of the recommended
methods. The tissue powder is lysed in a microcentrifuge tube with an extraction buffer
containing the detergent SDS or CTAB. After chloroform extraction and centrifugation,
gDNA is precipitated from the supernatant using isopropanol or alcohol.
Materials
Rice leaf tissue
Tungsten carbide beads (diameter 4 mm or 3 mm)
Liquid nitrogen
Extraction buffer 1 (see recipe; containing SDS) or extraction buffer 2 (see recipe;
containing CTAB)
Protein-denaturation mixture (see recipe)
3 M sodium acetate, pH 5.2
Isopropanol
70% ethanol
TE buffer, pH 8.0
10 mg/ml RNase A (store at 20C)
Additional reagents and equipment for checking DNA concentration and quality
(Basic Protocol 1, steps 11 to 13)
Grinding of plant tissue
There are three methods (steps 1a, 1b, or 1c) to grind plant tissue, depending on the
need.
1a. Put about 0.5 to 0.6 g rice leaf tissue into a 2-ml microcentrifuge tube, add a tungsten
carbide bead, cover the cap, and soak in liquid nitrogen for 1 min. Then, grind the
tissue with grinder such as FastPrep-24 Grinder or CK1000D Grinder (about 30 sec)
according to the instruction. Rapidly pour out the bead from the tube.
If a grinding machine like FastPrep-24 or CK1000D Grinder is available, this method is
the most efficient.
1b. Grind about 0.5 to 0.8 g fresh or frozen rice leaf tissue to a fine powder in a mortar
in liquid nitrogen, using a pestle. Transfer the powder (about 0.3 to 0.5 g, with a
little bit of liquid nitrogen) into a 2-ml microcentrifuge tube. If multiple samples
are treated, temporarily store the tube(s) with tissue powder in a freezer (or a box
containing liquid nitrogen) when grinding other samples.
1c. Alternatively, put about 0.5 to 0.6 g rice leaf tissue into a 2-ml centrifuge tube (on
a tube rack), add liquid nitrogen, then use a Phillips screwdriver to grind the leaves
into powder, occasionally adding more liquid nitrogen. Wash the screwdriver with
water in a beaker between samples.
To get a high efficiency for preparation of large number of samples, simultaneously work Preparation
with 24 or 30 samples according to the microcentrifuge rotor to be used. of Plant gDNA
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Current Protocols in Plant Biology Volume 1
Extraction and purification of DNA
2. Before thawing of the powder, add 800 l extraction buffer 1 or buffer 2 (preheated
to 65C), mix well by inversion or on a shaker, then incubate (the tubes and the rack)
in a 65C water bath for 20 to 30 min, with occasional inversion of the tubes.
If less tissue is used, add about 200 l extraction buffer per 100 mg tissue. For normal
tissues, use extraction buffer 1. For tissues rich in polysaccharides and for higher DNA
purity, use extraction buffer 2.
3. Add 800 l protein-denaturation mixture to each tube and mix well by inversion.
Then, shake the tubes gently in a shaker (about 50 to 60 rpm) for 10 min.
4. Microcentrifuge the mixture 10 min at 8,000 to 10,000 rpm, 4C. Transfer the
supernatant (about 600 l) into a 1.5-ml centrifuge tube.
If the supernatant is still turbid, repeat step 3 using 600 l protein-denaturation mixture.
5. For supernatant using extraction buffer 1, add 0.1 vol 3 M sodium acetate, pH 5.2,
to the supernatant, and mix well.
For supernatant using extraction buffer 2, there is no need to add sodium acetate.
6. Add 0.65 vol isopropanol, mix well by inversion, and leave at 20C for 1 hr.
7. Microcentrifuge 5 to 10 min at 10,000 rpm, 4C. Carefully pour out the supernatant
and remove residual liquid with a pipet, or invert the tubes.
8. Wash the DNA pellet twice with 70% ethanol, remove any liquid ethanol, and
slightly air-dry the pellet.
Do not overly dry the pellet, otherwise it will be difficult to dissolve in TE buffer.
9. Add 100 l TE buffer supplemented with 10 /ml RNase A to dissolve the DNA,
heating at 50 to 60C and vortex several times, each time for a few seconds.
10. Check the DNA concentration and quality as described in steps 11 to 13 of Basic
Protocol 1.
9. Pour the nuclei/agarose gel mixture into a gel mold with the wide-pore pipet into a
gel mold, and leave the mold on ice for 10 min.
Lysis of nuclei and protein degradation
10. Transfer the gel plugs carefully into a 50-ml centrifuge tube, add 10 ml (5 vol)
lysis buffer, and gently shake the tube at 50C for 12 hr. Change lysis buffer and Preparation
incubate at 50C for another 12 hr. of Plant gDNA
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Current Protocols in Plant Biology Volume 1
Figure 1 Purification of megabase gDNA by pulsed-field gel electrophoresis (PFGE). A low-
melting agarose gel is set into a normal agarose gel in 0.5 TBE electrophoresis buffer. The gel
plugs containing megabase gDNA are adhered to the low-melting agarose gel, and the gDNA is
run into the gel by pulsed field electrophoresis (with 0.5 TBE) using a BioRad CHEF Mapper XA
apparatus at 5 V/cm and 14C with switching time of 50 sec for 6 to 8 hr. After electrophoresis,
a part of the gel is cut out containing about 3 mm width of the DNA, this is stained in a DNA-
dye solution for 1 hr, and the DNA band position in the gel is marked by cutting. Based on the
positioning, the unstained gel slices (thickness of 3 to 4 mm) containing the gDNA are recovered.
11. Treat the gel plugs twice with 10 ml TEN buffer supplemented with 1 mM phenyl-
methanesulfonyl fluoride (PMSF) at 50C, each time for 30 min, then wash with
several changes of TEN buffer. Store the gel plugs in TEN buffer at 4C.
Alternatively, PMSF can be omitted, but wash the gel plugs with TEN buffer additional
times.
8. Use a 96-pin replicator to transfer small amounts (0.5 to 1.0 l for each pin) of
the crude gDNA solutions (step 6) into the 96-well PCR plates containing the PCR
mixture.
If use an 8- or 12-channel pipettor to transfer the template gDNAs to the PCR reactions,
add 150 l deionized water (NOT TE or preparation buffer) to dilute the gDNA solution,
and take 1.5 l each (no more than 2 l) to use in PCR.
9. Perform PCR cycling (Kramer and Coen, 2000) with a suitable thermal-cycling
program, e.g.,
Analyze the amplified products by PAGE (<300 bp; see Gallagher, 2012) or agarose
gel electrophoresis (<300 bp; see Voytas, 2000).
Washed 96-well deep-well plates can be reused for genotyping of any types of co-dominant
polymorphic markers. However, for analysis of samples with dominant markers, such as
testing the presence/absence of a specific DNA sequence, new 96-well deep-well plates
should be used to avoid false positive due to the trace contamination. For dominant
markers, setting a negative control reaction is necessary.
High-salt TE buffer
10 mM TrisCl pH 8.0
1 mM disodium EDTA
1 M NaCl (store at room temperature)
Store up to 1 month at room temperature
Lysis buffer
10 mM TrisCl pH 8.0
0.45 M disodium EDTA, pH 8.0
1% (w/v) Sarcosyl
0.5 mg/ml proteinase K (add just before use)
Store up to at least 2 years at room temperature without adding proteinase K
NIB
10 mM TrisCl, pH 9.5
10 mM disodium EDTA, pH 8.0
100 mM KCl
0.5 M sucrose
Store with the above ingredients up to 6 months at 4C
4.0 mM spermidine (add just before use)
1.0 mM spermine (add just before use)
0.1% (v/v) mercaptoethanol (add just before use)
NIB/Triton X-100
Prepare NIB (see recipe) supplemented with 10% (v/v) Triton X-100; dissolve Triton
X-100 completely. Store up to 1 week at 4C.
Preparation buffer, 10
100 mM TrisCl, pH 9.5
5 mM disodium EDTA
1 M KCl
Preparation
Store up to 1 month at room temperature of Plant gDNA
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Current Protocols in Plant Biology Volume 1
Protein-denaturation mixture
76 parts (v/v) chloroform
4 parts (v/v) isoamyl alcohol
20 parts (v/v) ethanol
Keep in a dark place or a dark container and close lid tightly to avoid evaporation
of chloroform
Prepare fresh at time of use
TE buffer
10 mM TrisCl, pH 8.0
1 mM disodium EDTA
Store up to 1 month at room temperature
TEN buffer
10 mM TrisCl, pH 8.0
1 mM disodium EDTA
30 mM NaCl
Store up to 1 month at room temperature
COMMENTARY
Background Information oughly as possible. To isolate high-molecular
According to different purposes and sit- weight gDNA, it is important to avoid thaw-
uations, various methods have been devel- ing of frozen tissue and ground powder before
oped for the preparation of plant gDNA. The adding the extraction buffer. Otherwise, the
CTAB method (Basic Protocol 1) is widely released cellular nucleases may degrade the
used for large-scale gDNA preparation and can gDNA. In addition, lysates (Basic Protocols
yield high-purity gDNA. The mini-preparation 1 and 2) and DNA solution should be treated
method (Basic Protocol 2) is much easier gently to minimize shearing of the gDNA.
and the most widely used. When the size During nuclei isolation (Basic Protocols 3),
of the gDNA is the primary concern, the the nuclei isolation buffer (NIB) should be kept
nuclear DNA preparation introduced in Ba- ice-cold until step 8. When recovering the nu-
sic Protocol 3 is capable of isolating pure clei suspension and the large-size gDNA af-
nuclear gDNA larger than 2.5 Mb, which ter digesting the low-melting agarose gel by
fulfills the needs of the majority of manipula- -agarase, a wide-pore pipet should be used to
tions requiring very long DNA, such as large- reduce shearing force to the nuclei and gDNA.
insert cloning and DNA-fiber FISH (fluores- Although the gDNA concentrations pre-
cence in situ hybridization). Meanwhile, for pared by Basic Protocol 4 are low (1 to
DNA marker-based genotyping in map-based 3 ng/l), use of 0.2 to 1 l of the solution
cloning or molecular breeding, which often as a template produces good PCR amplifica-
needs to deal with large numbers of samples, tion for DNA markers and small fragments
Basic Protocol 4 fulfills the requirement for (<2 kb). On the other hand, in this protocol it
high-throughput at very low cost. is important to control the amount of broken
tissue (2 to 5 mg) in a given volume of buffer
Critical Parameters (150 l). The starting tissue that can be broken
Various types of tissue, including whole should not exceed 10 mg, since gDNA solu-
seedlings, leaves, cotyledons, seeds/grains, en- tion produced from excessive tissue may bring
dosperm, embryos, and callus, can be used in more impurities that inhibit the activity of
for gDNA preparation. In most cases, fresh or Taq enzyme. In addition, adding more than 1.5
frozen leaves from young seedlings are prefer- l of the gDNA solution to a PCR (15 to 20
able, although dried tissues can also be used to l) can introduce more impurities and EDTA,
extract gDNA. Old tissues are rich in polysac- which decrease the amplification efficiency.
charides, which are difficult to separate from
gDNA.
To obtain a higher yield of gDNA (Basic Troubleshooting
Preparation Protocols 13), plant tissue should be ground Table 2 lists some problems that may
of Plant gDNA
in liquid nitrogen to a fine powder as thor- be encountered with the protocols described
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Volume 1 Current Protocols in Plant Biology
Table 2 Troubleshooting for gDNA Preparations and Applications
in this article, along with accompanying the gDNA in the gel plugs is 100 ng/l. Af-
solutions. ter re-purification by electrophoresis on a low-
melting agarose gel, a portion of the gDNA
Anticipated Results may be lost, and the concentration of the
gDNA in the gel slices may be 50 ng/l.
Maxi CTAB gDNA preparation
This method yields about 500 to 1000 g
gDNA of high purity from 5 g leaf tissue, with
sizes >50 kb. High-throughput gDNA preparation
Using gDNA templates prepared with this
Mini gDNA preparation method, PCRs with diagnostic marker primers
About 50 to 100 g gDNA can be obtained (such as SSRs and indels) reach more than
from 0.5 g fresh tissue in each preparation. 95% success rates.
Most DNA fragments from this method are
larger than 20 kb.
Time Considerations
Nuclear DNA preparation
About 200 to 300 g of nuclear DNA of Maxi CTAB gDNA preparation
>2.5 Mb in size can be obtained from 20 to This procedure should take 2 to 3 hr for Preparation
of Plant gDNA
30 g of young leaf tissue. The concentration of working with 4 to 8 samples simultaneously.
41
Current Protocols in Plant Biology Volume 1
Mini gDNA preparation electrophoresis of proteins. Curr. Protoc. Mol.
This procedure should take 1.5 to 2 hr for Biol. 97:10.2A.1-10.2A.44.
working with 24 to 30 samples simultaneously. Gemmill, R.M., Bolin, R., Albertsen, H., Tomkins,
J.P. and Wing, R.A. 2002. Pulsed-field gel elec-
Nuclear DNA preparation trophoresis for long-range restriction mapping.
This procedure should take 2 to 3 hr for Curr. Protoc. Hum. Genet. 31:5.1.1-5.1.27.
working with 1 to 2 samples simultaneously. John, M.E. 1992. An efficient method for isola-
tion of RNA and DNA from plants containing
High-throughput gDNA preparation polyphenolics. Nucleic Acids Res. 20:2381. doi:
The time for one round of preparation of 96 10.1093/nar/20.9.2381.
samples mainly depends on the speed of col- Kramer, M.F. and Coen, D.M. 2000. Enzymatic
lecting seedling leaf fragments into the 96-well amplification of DNA by PCR: Standard proce-
dures and optimization. Curr. Protoc. Mol. Biol.
deep-well plate. The processes of adding beads 56:15.1.1-15.1.14.
and preparation buffer, and breaking with a
Liu, Y.G. and Whittier, R.F. 1994. Rapid preparation
vortex shaker, take about 6 to 8 min. Use of of megabase plant DNA from nuclei in agarose
a home-made device to add 96 beads simul- plugs and microbeads. Nucleic Acids Res.
taneously into a 96-well deep-well plate can 22:2168-2169. doi: 10.1093/nar/22.11.2168.
further speed up the process. Lodhi, M.A., Ye, G.N., Weeden, N.F., and Resisch,
B.I. 1994. A simple and efficient method for
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Preparation
of Plant gDNA
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