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Preparation of Rice Plant Genomic DNA

for Various Applications


Lian Zhou,1,2,3 Rongxin Shen,1,2,3 Xingliang Ma,1,2,3 Heying Li,1,2,3
Gousi Li,1,2,3 and Yao-Guang Liu1,2
1
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,
Guangzhou, China
2
College of Life Sciences, South China Agricultural University, Guangzhou, China
3
These authors contributed equally to this work

Research on plant molecular biology, genetic engineering, and crop molecular


breeding involves various manipulations of plant genomic DNAs (gDNAs).
These studies require preparing gDNAs from plant materials using suitable
methods according to the yield, intactness, and purity of the resultant gDNA
samples. Here we describe protocols for preparation of rice plant gDNAs for
various applications including: (1) maxipreps and (2) minipreps of gDNAs
for restriction analysis, gene cloning, PCR amplification, genomic sequencing,
and genotyping; (3) preparation of megabase-sized nuclear DNA for construc-
tion of large-insert genomic libraries and long-range physical mapping; and
(4) 96-well-format high-throughput gDNA preparation for PCR-based geno-
typing. The methods are also suitable for other plant species including dicots.
C 2016 by John Wiley & Sons, Inc.

Keywords: DNA preparation r genomic DNA r nuclear DNA r plants

How to cite this article:


Zhou, L., Shen, R. Ma, X., Li, H., Li, G. and Liu, Y.-G. 2016.
Preparation of rice plant genomic DNA for various applications.
Curr. Protoc. Plant Biol. 1:29-42.
doi: 10.1002/cppb.20002

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.

The CTAB (hexadecyltrimethylammonium bromide) DNA preparation method is suit-


able for preparation of a large amount of high-quality gDNA (Murray and Thompson,
1980; Richards et al., 1994). The resultant gDNA is suitable for Southern blot analysis,
PCR amplification of large fragments, genomic library construction, and next-generation
sequencing. For most applications such as restriction analysis, PCR amplification of
DNA fragments, PCR-based genotyping, and next-generation sequencing, a simpler
SDS (sodium dodecyl sulfate) method (Dellaporta et al., 1983) and modified CTAB
method (Doyle and Doyle, 1987) can be used for minipreps of gDNA. In the case
of large-scale genotyping with DNA markers, e.g., SSRs (simple sequence repeats),

Preparation
of Plant gDNA

Current Protocols in Plant Biology 1:29-42, May 2016 29


Published online May 2016 in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cppb.20002 Volume 1
Copyright C 2016 John Wiley & Sons, Inc.
Table 1 A Summary of the Four Basic Protocols for Plant gDNA Preparation

Basic Protocol 1 Basic Protocol 2 Basic Protocol 3 Basic Protocol 4


Time course 2-3 hr 1.5-2 hr 2-3 hr 6-8 min (96 samples)
Throughput Low High Low Very high
Tissue amount 2-5 g 0.5 g 20-30 g 2-5 mg
DNA length >50 kb >20 kb >2.5 Mb <10 kb
Storage period Long Long Long Short
Yield 100-200 g/g 50-100 g 200-300 g 1-3 ng/l (150 l)
tissue
Cytoplasmic DNA Included Included Excluded Included
Application PCR, genomic PCR, DNA Large-insert PCR, DNA markers
cloning, markers, cloning,
restriction, restriction, DNA-fiber FISH
Southern blotting genomic cloning

indels (insertions/deletions), and SNPs (single nucleotide polymorphisms), preparation


of gDNA templates from massive samples is time-consuming and tedious. Therefore,
a very high-throughput gDNA preparation method has been developed to deal with
large number of samples for PCR-based genotyping (Wang et al., 2013). Very-large-size
gDNA is necessary for large-insert genomic library construction and long-range physical
mapping. Since the chromosomal DNA molecules are extremely fragile in liquid buffer,
embedding the isolated nuclei into low-melting point agarose gel plugs can provide
gDNA of megabase size (Liu and Whittier, 1994).

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.

BASIC PREPARATION OF PLANT GDNA USING CTAB


PROTOCOL 1
This protocol describes a gDNA extraction method using the amine-based cationic qua-
ternary surfactant CTAB in the extraction buffer for lysing cells and denaturing proteins.
CTAB can form a soluble complex with nucleic acids under high-salt conditions (>0.7 M
NaCl), and precipitate DNA at lower salt concentrations (<0.5 M). This method is suit-
able for large-scale DNA extraction and yields high-quality gDNA, which can be used for
cloning, Southern blot hybridization, and other manipulations that require high-quality
Preparation
of Plant gDNA DNA.

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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)

Mortars and pestles


50-ml centrifuge tubes and tube rack
55 and 65C water baths
Shaker
Centrifuge (Eppendorf 5810R) and angle rotor (F-34-6-38, with adapters) for
50-ml centrifuge tubes
Spectrophotometer (NanoDrop2000, Thermo Scientific)
Additional reagents and equipment for agarose gel electrophoresis (Voytas, 2000)
Grinding of plant tissue
1. Grind about 4 to 5 g rice plant tissue for each sample into a fine powder with a
mortar and a pestle in the presence of liquid nitrogen. Transfer the frozen tissue
powder to an organic solventresistant 50-ml centrifuge tube and temporarily store
in a freezer when grinding other samples.
Work with even numbers of samples for each round of gDNA preparation, to facilitate
centrifugation.
Use young tissue (leaves) and avoid larger stems and veins to achieve higher gDNA
yields with minimal contamination of polysaccharide.
If samples need to be stored in a freezer, it is desirable to first freeze the samples with
liquid nitrogen in advance.
Avoid thawing the samples during storage and before adding extraction buffer in the
gDNA preparation.

Extraction and purification of gDNA


2. Add 20 ml (or 4 ml per g tissue) CTAB extraction buffer (preheated to 80C) and
mix thoroughly by inversion. Incubate the lysate mixture at 65C for 15 to 20 min,
with occasionally shaking.
3. Add 15 ml (or 0.75 times the volume of the added extraction buffer) of 24:1 (v/v)
chloroform/isoamyl alcohol to each tube and mix well by inversion. Set the tubes in
a slanted position in a shaker and shake at about 50 rpm for 10 min. Centrifuge at 15
min at 12,000 g (8,000 rpm in Eppendorf F-34-6-38 rotor), room temperature.
Be sure to balance the tubes when adding chloroform/isoamyl alcohol.

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)

1.5-ml and 2-ml microcentrifuge tubes and tube rack


FastPrep-24 Grinder (MP, Biochemicals) or CK1000D Grinder (Thmorgan)
Mortars and pestles
Phillips screwdriver
65C water bath
Shaker
Microcentrifuge

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.

BASIC PREPARATION OF MEGABASE PLANT GDNA


PROTOCOL 3
This protocol describes a method to prepare megabase plant nuclear DNA. Nuclei are
released from ground leaf tissue in nuclei isolation buffer (NIB), in which chloroplasts are
lysed by the non-ionic surfactant Triton X-100. Nuclei are collected by centrifugation
and embedded in low-melting agarose gel plugs. The nuclei are lysed with the ionic
surfactant Sarcosyl and chromatin proteins are degraded by proteinase K. The nuclear
gDNA in low-melting agarose gel can be used for restriction digestion for genomic
library construction and other applications.
Materials
Seeds of interest
Liquid nitrogen
NIB/Triton X-100 (see recipe)
Nuclei isolation buffer (NIB; see recipe)
1.8% (w/v) low-melting agarose prepared in 45 mM Tris borate/1 mM disodium
EDTA (pH 8.0)
Lysis buffer (see recipe)
Phenylmethanesulfonyl fluoride (PMSF)
TEN buffer (see recipe)
Preparation
of Plant gDNA 28 to 30C growth chamber for seeds
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Volume 1 Current Protocols in Plant Biology
Mortar and pestle
500-ml glass beakers
Metal medicinal spoon
Nylon meshes with pore sizes of 149 m, 74 m, and 37 m
50-ml centrifuge tubes
Centrifuge (Eppendorf 5810R) and an angle rotor (F-34-6-38) with adapters for
50-ml centrifuge tubes
45 and 50C water baths
Mold for agarose gel plugs (BioRad or home-made)
Shaker
CHEF Mapper AX apparatus (BioRad)
Additional reagents and equipment for pulsed-field gel electrophoresis (Gemmill
et al., 2002) and agarose gel electrophoresis (Voytas, 2000)
Plant seedling growth
1. Sow about 2,000 to 3,000 seeds (for example, rice) on several layers of tissue paper
in a tray and grow in a growth chamber at 28 to 30C under 12-hr light/day for
10 days, then in dark for 5 days. For dicot species, grow plants at appropriate
temperature under 12-hr light/day for 15 to 20 days, and in dark for 5 days.
Green seedlings also can be used, but use of etiolated seedlings can reduce chloroplast
content.
Isolation of nuclei
2. Collect 25 g leaf tissue (20 g for dicot plants; can be frozen with liquid nitrogen and
stored in a 80C freezer), and grind to fine powder with a mortar and pestle.
3. Transfer the powder to a 500-ml glass beaker set on ice. After the liquid nitrogen
evaporates completely (do not allow thawing), add 200 ml ice-cold NIB, and mix
gently with a medicinal spoon.
4. Filter the homogenate sequentially through one layer each of 149 m, 74 m, and
37 m nylon meshes into a 500-ml beaker set on ice.
5. Add 1/20 vol of NIB/Triton X-100 (chilled on ice) to the filtrate and mix gently.
6. Transfer the filtrate into 50-ml tubes, then centrifuge 10 min at 1,200 g, 4C, to
pellet the nuclei.
7. Resuspend the nuclei with 10 ml NIB in each tube, collect all nuclei suspension into
one tube, and centrifuge 10 min at 1,200 g, 4C. After removing the supernatant,
first add about 0.5 ml (or less) NIB and gently resuspend the nuclei using a 1-ml wide-
pore pipet (prepared by cutting 3 to 4 mm off of the tip). Adjust the nuclei suspension
by adding a little NIB, using the wide-bore pipet, to a final volume of 1.2 to 1.3 ml.
8. Set the tube (nuclei) in a water bath at 45C for 2 to 3 min, add 0.5 vol 1.8% low-
melting point agarose (heated to 50C) using the wide-bore pipet, then mix well by
stirring gently (final volume, 1.8 to 2.0 ml).
Keep the tube in the water bath while mixing.

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.

Test of DNA quality


12. Cut a small piece (5 mm 5 mm) of the plug and prepare a pulsed field gel
electrophoresis (using a 1% agarose/0.5 TBE gel) with a yeast chromosome size
standard, using a CHEF Mapper AX apparatus at 5 V/cm and 14C with switching
times of 70 sec for 12 hr and 100 sec for 12 hr.
The gel plugs can be directly used for restriction digestion for genomic library con-
struction; alternatively, the gDNA can be further purified to high quality as described in
step 13.
Protocols for pulsed-field gel electrophoresis are provided in Gemmill et al. (2002)

Re-purification of the gDNA


13. Optional: Run the gDNA into a low-melting agarose gel (Voytas, 2000) and recover
the gel slices containing the gDNA as indicated in Figure 1. Transfer the gel slices
into a 50-ml centrifuge tube, wash twice in TEN for 1 hr each time, then remove
TEN buffer and store the gel plugs at 4C.

BASIC HIGH-THROUGHPUT PREPARATION OF PLANT GDNA FOR PCR-BASED


PROTOCOL 4 GENOTYPING
This protocol describes a rapid and high-throughput method for preparation of plant
gDNA for PCR. A 96-well format is adopted for preparing gDNA and transferring the
DNA templates to PCR reactions. In this method, total gDNAs of 96 samples are released
into the preparation buffer by breaking the tissue in a 96-well deep-well plate. Without the
Preparation lengthy DNA purification and ethanol-precipitation processes, the crude gDNA solutions
of Plant gDNA
are directly applied to 96 PCRs. Therefore, this method is high-throughput and especially
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Volume 1 Current Protocols in Plant Biology
suitable for genotyping large numbers of plant samples using PCR markers. The prepared
template gDNAs also can be used for amplification of relatively large DNA fragments
(1 to 2 kb).
Materials
Seeds of interest
Soil
Tungsten carbide alloy beads (diameter of 4 mm or 3 mm) with high specific gravity
10 preparation buffer (see recipe)
Taq DNA polymerase and buffer
dATP, dCTP, dGTP, and TTP
Appropriate forward and reverse primers
96-well deep-well plates (2.2-ml well size) and silicone rubber covers
8- or 12-channel pipettor
Centrifuge (Eppendorf 5810R) and basket rotor for 96-well plates
96-pin replicator (a commercial product, or a home-made one using #304 stainless
steel wire of 1.2-mm diameter)
Additional reagents and equipment for PCR (Kramer and Coen, 2000),
polyacrylamide gel electrophoresis (PAGE; Gallagher, 2012), and agarose gel
electrophoresis (Voytas, 2000)
Plant seedling growth
1. Prepare a bottomless plate by removing the bottom, by cutting or puncturing, of a
96-well PCR plate or a 96-well deep-well plate (1.6-ml or 2.2-ml). Fill the wells of
the bottomless plate with soil (if a PCR plate is used, no soil is added), and grow 96
monocot or dicot seedlings to a three- to five-leaf stage.
Preparation of template gDNAs
2. For monocot seedlings, take an appropriate length (about 4 cm, equal to the depth of
the 2.2-ml wells of a 96-well deep-well plate) of a leaf fragment from a seedling and
place into each well of 2.2-ml 96-well deep-well plate. For dicot plants, take a small
piece (2 to 4 mg and do not exceed 8 mg) of leaf and place into each well.
The weights of several small leaf pieces of dicot plants can be measured first using an
analytical balance; other samples can then be estimated by experience.
Putting a 4-cm long of monocot plant leaf fragment rather than a short piece of 5 to
6 mm (equal to 2 to 4 mg) into a well has the advantage that the bottom part of the leaf
fragment always can be broken by the beads; while the short leaf piece may attach on the
well wall during vortexing (see step 5, below).
3. Put a tungsten carbide alloy bead in each well.
Beads of 4-mm diameter are better than those of 3-mm diameter. Ordinary steel beads
have less specific gravity, and are thus not effective for breaking the tissue. In addition,
they rust easily; rust may inhibit the activity of Taq enzyme.
4. Add 150 l of 1 preparation buffer to each well using a 12 (or 8)-channel pipettor.
Seal the plate with a layer of plastic wrap and a silicon rubber cover.
Alternatively, 0.5 TE buffer also can be used as the preparation buffer.
5. Vigorously shake the 96-well deep-well plate with a vortex mixer for 2 to 4 min until
the buffer become green. For breaking small leaf pieces of dicot plants, occasionally
tap the plate to dislodge the leaf pieces that attach to the well wall so that they fall to
the bottoms of the wells.
Generally, only the bottom part (4 to 6 mm, 2 to 4 mg) of the 4-cm long leaf fragments Preparation
of Plant gDNA
can be broken by the beads.
37
Current Protocols in Plant Biology Volume 1
6. Centrifuge the plates 10 sec at 1500 rpm in an Eppendorf 5810R rotor with a 96-welll
plate carrier at room temperature.
After a brief centrifugation, the prepared crude gDNA solutions containing 1 to 3 ng/l
gDNA can be directly used as PCR templates.
The crude gDNA solutions can be stored at 20C for later use. In this case the beads can
be taken out for reuse using a strong magnet; wrapping the magnet with layers of tissue
paper facilitates taking out the beads from the magnet.

Application for PCR-based genotyping


7. Prepare 1.6 ml of PCR mix for each 96-well PCR plate, including:

40 to 45 U Taq DNA polymerase (every 35- to 40-l reaction contains 1 U)


100 to 150 M each of dATP, dCTP, dGTP, and dTTP
0.2 M each primer.
Add 15 to 16 l of mix to each well of a 96-well PCR plate using an 8- or 12-channel
pipettor.
For amplification of DNA fragments of 1 to 2 kb, the PCR reaction is set at 25 to 30 l
with 1 U Taq DNA polymerase.
If two PCR markers are distinguishable by gel electrophoresis, they can be amplified in
the same reaction using the four primers.

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.,

1 cycle: 2 min 95C (initial denaturation)


3335 cycles: 20 sec 94C (denaturation)
30 min 55-60C (annealing)
20 sec 72C (extension).

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.

REAGENTS AND SOLUTIONS


Use deionized, distilled water in all recipes and protocol steps.

CTAB extraction buffer


1.5% (w/v) CTAB
75 mM TrisCl, pH 8.0
1 M NaCl
Preparation 15 mM disodium EDTA
of Plant gDNA Store up to 1 year at room temperature
38
Volume 1 Current Protocols in Plant Biology
CTAB precipitation buffer
1% (w/v) CTAB
50 mM TrisCl, pH 8.0
10 mM disodium EDTA (store at room temperature)
Store up to 1 year at room temperature
Extraction buffer 1
0.5 M NaCl
100 mM TrisCl, pH 8.0
0.05 M disodium EDTA
1.25% (w/v) sodium dodecyl sulfate (SDS)
Store up to 2 years or longer at room temperature
Extraction buffer 2
2% hexadecyltrimethylammonium bromide (CTAB)
1.4 M NaCl
20 mM disodium EDTA
100 mM TrisCl, pH 8.0
Store up to 2 years or longer at room temperature

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

39
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
40
Volume 1 Current Protocols in Plant Biology
Table 2 Troubleshooting for gDNA Preparations and Applications

Problem Possible cause Solution


Low yield of gDNA Old tissue Use young tissue if possible.
Tissue was not homogenized Grind tissue to fine powder as thoroughly as possible
completely
DNA pellet was incompletely Do not allow gDNA to dry completely. Resolve
dissolved gDNA at 65C to achieve completely dissolution.
gDNA is degraded Fresh sample was not immediately Sample should be frozen as soon as possible
frozen after collection
Sample thawed before adding The sample must be kept frozen at all times during
extraction buffer storage and grinding; liquid nitrogen should be added
promptly before finishing grinding
Contamination with Interphase and organic phases were Do not attempt recovering the aqueous layer near the
proteins or RNA pipetted up interphase
Insufficient chloroform extraction Repeat one more chloroform extraction until the
supernatant is clear
RNase A is not enough Add more RNase A
gDNA is resistant to gDNA solution contains high Re-purify gDNA as above
restriction digestion amount of proteins and other
impurities
gDNA solution contains a high After ethanol precipitation, wash the pellet
level of salt or residual ethanol completely with 70% ethanol and remove any liquid
ethanol; air-dry the pellet but do not overly dry it
Failure of PCR related Too much gDNA is applied to PCR Re-estimate the gDNA concentration by agarose gel
to gDNA template electrophoresis
Use reduced amount of gDNA as template (for gDNA
from Basic Protocols 1 and 2, use 10-30 ng per PCR)
For gDNA from Basic Protocol 4, if the gDNA
solution is dark green, this indicates that too large an
amount of tissue was applied. Dilute the solution
appropriately with deionized water, or re-prepare
gDNA using less tissue.

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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F., Sperisen, C., Vornam, B., and Ziegen- Murray, M.G. and Thompson, W.F. 1980. Rapid
hagen, B. 1998. Comparative analysis of dif- isolation of high molecular weight plant
ferent DNA extraction protocols: A fast, uni- ONA. Nucleic Acids Res. 8:4321-4326. doi:
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studies. Plant Mol. Biol. Rep. 16:69-86. doi: Preparation of genomic DNA from plant tis-
10.1023/A:1007428009556. sue. Curr. Protoc. Mol. Biol. 27:2.3.1-2.3.7. doi:
Dellaporta, S.L., Wood, J., and Hicks, J.B. 10.1002/0471142727.mb0203s27.
1983. A plant DNA minipreparation: Ver- Voytas, D. 2000. Agarose gel electrophoresis. Curr.
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Wang, H., Chu, Z., Ma, X., Li, R., and
Doyle, J.J. and Doyle, J.L. 1987. A rapid DNA Liu, Y.G. 2013. A high through-put proto-
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Preparation
of Plant gDNA

42
Volume 1 Current Protocols in Plant Biology

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