Growing Better Results
Automated Genomic DNA Purification Using the Wizard® Magnetic 96 DNA Plant System
B y S u s a n Ko l l e r, B . S . , H e m a n t h S h e n o i , P h . D. , J u d i t h B u r n h a m , M . B . A . , a n d R e x B i t n e r, P h . D. , P r o m e g a C o r p o r a t i o n

Abstract
The use of molecular biology techniques is rapidly expandingin basic and applied plant and agricultural research. Molecularanalysis has become the method of choice for large-scale markerassisted breeding, seed-quality testing, SNP discovery andscoring, and analysis of transgenic plants. Automated purificationof high-quality DNA from plant seed and leaf is essential to theseemerging methods. Here we introduce the highly consistent andautomated Wizard® Magnetic 96 DNA Plant System.

length polymorphism (AFLP; 2,3) and simple sequence
repeat/short tandem repeat analysis (SSR/STR; 4). The useof MagneSil™ Paramagnetic Particles(a) (PMPs) in this system offers highly consistent yield and ease of handling ineither manual or automated formats. Protocols are available
for the Biomek® 2000 Robotic Workstation (BeckmanCoulter) as well as other robotic platforms. Send request for
information about automated protocols to:plantbio@promega.com.

The use of MagneSil™ Paramagnetic Particles(a)(PMPs) in this system offers highly consistentyield and ease of handling in either manual orautomated formats.

Place fresh leaf or seed sample
in 96 deepwell plate in the presenceof Lysis Buffer A. Add 1 or 2
grinding beads.
Grind. Centrifuge.

Introduction
Transfer supernatant to a cleanplate. Add Lysis Buffer B/MagneSil™
PMPs mixture and mix well.
Incubate.

The purification of plant genomic DNA is important for
many applications in plant molecular biology. As largescale plant genomics and plant breeding projects are undertaken, there is an increasing need for automated highthroughput purification of genomic DNA from a wide variety of plant species. Analysis of plants during markerassisted breeding or the insertion of transgenic elements tomake genetically modified plants may require the use ofrobotic platforms to facilitate the purification of hundredsof thousands of samples per year.

Place the plate on the MagnaBot®
96 Magnetic Separation Device.
Discard liquid.

Add Wash Buffer and mix.
Place the plate on the MagnaBot®
96 Magnetic Separation Device.
Discard liquid. Repeat the wash.
Dry the particles.

Plant scientists often use “homebrew” methods to purify
genomic DNA from plant material. These methods can belabor- and time-intensive, often involving several centrifugation and incubation steps. For example, CTAB (hexadecyltrimethylammonium bromide) extraction is an accepted
methodology with many described variations for different
seed and leaf materials (1). This method generally involves
lysis and detergent solubilization of plant material in a
CTAB-containing buffer followed by organic extraction andalcohol/salt precipitation of DNA. Other techniques suchas differential lysis, salting out, and silica-filter plates orcolumns are not widely used. None of these methods arereadily adapted to automated robotic protocols.

Promega Notes

Remove purified DNA to a fresh plate.

3220MA01_1A

We designed the Wizard® Magnetic 96 DNA Plant System(a)(Cat.# FF3760, FF3761) to provide high-quality DNA for
downstream applications such as PCR(b), random amplification of polymorphic DNA (RAPD), amplified fragment

Add Nuclease-Free Water.
Place the plate on the MagnaBot®
96 Magnetic Separation Device.

Figure 1. Schematic of DNA isolation using the Wizard® Magnetic 96
DNA Plant System.

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Wizard ® Magnetic 96 Plant System ...continued
Results of DNA Isolation from Plants

M

Canola seedM

Sunflower seed

M

Tomato leaf

M

Corn leaf

MSoybeanM

Melon seed

Table 1. Times Required to Isolate DNA from Plant Tissue Using
Three Listed Procedures.
MethodStepsTimeWizard® Magnetic 96 DNA Plant System
1140 minutes
Commercial membrane plate
1755 minutes
CTAB homebrew method
16135 minutes

The key initial step in this procedure is grinding the plant
samples in a 96 well format. Plant tissue may be fresh,lyophilized or frozen in liquid nitrogen. Our data is from
fresh or frozen plant material ground at room temperatureusing a Geno/Grinder® mill (SPEX CertiPrep, Metuchen,NJ). A Retsch Grinding Mill (Haan, Germany) provides comparable results under similar conditions. Table 2 shows suggested grinding conditions for a variety of plant materials.
Table 2. Milling of Leaf and Seed Samples Using a SPEX
Certiprep Geno/Grinder® Mill.
Sample TypeConditions
Tender leaf or seedling
1 minute at 400 (1,200rpm)
Mature leaf
1–1.5 minutes at 500 (1,500rpm)
Fibrous leaf
1.5–2 minutes at 500 (1,500rpm)
Seeds
2–3 minutes at 500 (1,500rpm),
depending on size and seed coat.
May be repeated.

As illustrated in Figure 2, we isolated DNA from multiple
plant materials using the Wizard® Magnetic 96 DNA Plant
System and the automated protocol for the Biomek® 2000
robotic platform. The Wizard® Magnetic 96 DNA Plant
System consistently delivered high-molecular weight DNA
that was largely free of contaminating RNA.
DNA isolated using the Wizard® Magnetic 96 DNA Plant
System can be used for many downstream applications. Figure
3 shows PCR amplification of an intron from the TrnL chloroplast gene from a number of plant seed and leaf tissue DNAsamples isolated using the method described in Figure 1. For
some plant samples such as corn leaf, DNA yield is sufficientto perform at least 100 standard PCR amplifications. All samples showed strong amplification of the TrnL sequence. The263498TA07_1AThe Wizard® Magnetic 96 DNA Plant System protocol isoutlined in Figure 1. This flexible procedure allows thepurification of genomic DNA from a wide variety ofseed and leaf material with only minor modifications tothe protocol. When performed manually, the single centrifugation step combined with the ease of handling of
the MagneSil™ Paramagnetic Particles saves time overother methods (Table 1).

Figure 2. DNA isolated from different plant species using the Wizard®
Magnetic 96 DNA Plant System. DNA was isolated using the automatedprotocol available for the Biomek® 2000 instrument. The starting materialfor each lane was either 5 canola seeds, 1 sunflower seed, 8 tomato leaf
punches (8mm in diameter), 8 corn leaf punches (8mm in diameter),
20mg of soybean or 1 watermelon seed. Samples were eluted in 50µl
Nuclease-Free Water, and 15µl of eluate were run on a Latitude®
(BioWhittaker) 1% agarose TBE gel stained with ethidium bromide. Eightsamples were shown for each plant tissue to indicate reproducibility of themethod. DNA from the canola seed appears partially sheared due tomilling. Lanes M, Lambda DNA/Hind III Markers (Cat.# G1711).

diversity of amplification products is due to polymorphism
within these loci between plant species.
We have purified DNA from many different plant samples
using the Wizard® Magnetic 96 DNA Plant System anddemonstrated that the DNA could be used for PCR or
RAPD analysis (Table 3). Typical yields from several plantM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15bp

M

No PVPP

PVPP

1,500 –

3315TB03_1A

500 –

100 –
Tobacco seedling
Soybean
Lettuce leaf
Corn leaf
PotatoArabidopsis leaf
Cabbage seedGreen pepper seed

9101112131415Prairie grass, seed headChives
Tomato leaf
Rice
Canola seed
Sunflower seed
Carrot seed

3499TA07_1A

12345678

Figure 3. The Wizard® Magnetic 96 DNA Plant System produces PCRquality DNA from a variety of plant species. Shown are PCR products forchloroplast DNA using a universal primer pair that amplifies the intron of
the TrnL chloroplast gene across many plant species (5). The template
was 1µl of eluate. One or more bands are produced depending on theplant species.

Table 3. Plant Sample Types Processed Using the Wizard®
Magnetic 96 DNA Plant System.
Arabidopsis
Cotton seed
Soybean
Cabbage seed
Grass seedSquashCanola leaf
Green pepper seedSquash seed
Canola seed
Lettuce
Strawberry leaf*
Carrot seed
Milkweed leaf
Sunflower seed
Chicory leaf
Potato tuber
Tobacco seedling
ChivesRadish leaf
Tomato leaf
Corn leaf
Rice leaf
Tomato seed
Cotton leaf*
Sorghum
Watermelon seed
*These samples need the addition of PVPP to the lysis buffer to remove
phenolic compounds that inhibit PCR.

Table 4. Typical DNA Yield From Different Plant Species Using
the Wizard® Magnetic 96 DNA Plant System.
SampleYield
Arabidopsis tissue (50mg)
10ng/mg tissue
Canola leaf punches* (twelve)
26ng/leaf punch
Canola seeds (five)343ng/seedCorn leaf punches* (twelve)
98ng/leaf punch
Cotton seed (one)29ng/seed
Lettuce leaf punches* (eight)
13ng/leaf punch
Melon seed (one)166ng/seed
Radish leaf punches* (twelve)
89ng/leaf punch
Soybean (10mg)
10ng/mg bean
Squash seed (one)279ng/seed
Sunflower seed (one)405ng/seed
Tomato leaf punches* (twelve)
111ng/leaf punch*Leaf punches are 6mm in diameter.

Promega Notes

Figure 4. RAPD analysis of cotton leaf DNA isolated using the Wizard®
Magnetic 96 DNA Plant System in the presence or absence of PVPP. A
cotton leaf was homogenized using a SPEX CertiPrep Geno/Grinder® millin the presence or absence of 10mg PVPP/300µl Lysis Buffer A. DNA wasisolated manually from each leaf as described in Technical Bulletin #TB289.
RAPD analysis was performed on cotton leaf DNA using the Pharmacia
Ready-To-Go™ RAPD Analysis Kit. Samples were diluted 1:10 in water,and 1, 2, 3 or 4µl were used in a RAPD reaction with primer #3. Fivemicroliters of each sample were analyzed on a 1.2% E-Gel™ agarose gel(Invitrogen).

species and tissues are shown in Table 4. DNA was quantitated using PicoGreen® fluorescence (Molecular Probes,Eugene, OR).

PCR Amplification of Purified DNA and RAPD
Analysis of Cotton Leaf
To further illustrate the importance of high-quality DNA
for amplification, we analyzed DNA purified from cottonleaf. Cotton leaf contains high amounts of polyphenolics(6), which irreversibly bind to DNA during purification andcan lower yield as well as inhibit downstream analysis ofpurified DNA (7). Polyvinylpolypyrrolidone (PVPP) isthought to reduce copurification of phenolic compoundswith DNA that occurs during the initial crushing or grinding steps of the protocol (8).
Figure 4 shows RAPD analysis using DNA isolated from
cotton leaf with the Wizard® Magnetic 96 DNA Plant
System in the presence or absence of PVPP (e.g., Cat.#P6755, Sigma, St. Louis). Cotton leaf DNA purified without
PVPP showed limited amplification. Inhibition of the RAPD
reaction increased as increasing amounts of DNA sample
were used, suggesting the presence of inhibitors in the purified DNA. In contrast, the addition of PVPP to the Wizard®
Magnetic 96 DNA Plant System resulted in strong amplification of the expected diverse amplification products. The use

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Number 79 200127

Wizard ® Magnetic 96 Plant System ...continuedof PVPP with the Wizard® Magnetic 96 DNA Plant System
is a simple, cost-effective method that can be used in anautomated system or manually to isolate high-quality DNA
from tissue containing high levels of phenolic compounds.

Conclusions
The Wizard® Magnetic 96 DNA Plant System can be used
manually or with an automated workstation to consistently
isolate high-quality DNA from plant tissue. The flexible
design of this system allows the reliable purification of
DNA from widely diverse plant materials and sample
types. PVPP can be used with the Wizard® Magnetic 96
DNA Plant System to isolate DNA from highly phenolicplant samples.

Susan Koller, B.S.
R&D Scientist

Hemanth Shenoi,Ph.D.
Product Manager

Judith Burnham,M.B.A.
Marketing Manager

References1. Doyle, J.J. and Doyle, J.L. (1987) Phytochem. Bull. 19, 11–15.
2. Vos, P. et al. (1995) Nucl. Acids Res. 23, 4407–4414.
3. Zambeau, M. and Vos, P. (1993) European Patent Application
No. 92402629.7. Publication No. 0534858A1.
4. Edwards, A. et al. (1992) Genomics 12, 241.
5. Taberlet, P. et al. (1991) Plant Mol. Biol. 17, 1105–1109.
6. Zhang, J. and Stewart, J. McD. (2000) J. Cotton Sci. 4, 193–201.
7. Schneiderbauer, A., Sandermann, Jr., H. and Ernst, D. (1991) Anal.
Biochem. 197, 91–95.
8. Lodhi, M.A. et al. (1994) Plant Mol. Biol. Reporter 12, 6–13.

Protocol◆ Wizard® Magnetic 96 DNA Plant System Technical Bulletin #TB289,
Promega Corporation.
(www.promega.com/tbs/tb289/tb289.html)

Rex Bitner, Ph.D.
R&D Scientist

Ordering Information
ProductWizard® Magnetic 96 DNA Plant
System(a)Wash Buffer, Plant

Size

Cat.# Price ($)

2 × 96
4 × 9640ml

FF3760
FF3761A3811

Size
eacheach

Cat.# Price ($)
V8151
450
V8381
10334652
67

Related Products

factlabThe Biomek® 2000 instrument

can be used to automate DNAProductMagnaBot® Magnetic Separation Stand
MagnaBot® Spacer

MagnaBot and Wizard are trademarks of Promega Corporation and are registeredwith the U.S. Patent and Trademark Office. MagneSil is a trademark of Promega
Corporation.
Biomek is a registered trademark of Beckman Instruments, Inc. E-Gel is a trademark of Invitrogen. Geno/Grinder is a registered trademark of SPEX CertiPrep,
Inc. Latitude is a registered trademark of CBM Intellectual Properties, Inc.
PicoGreen is a registered trademark of Molecular Probes, Inc. Ready-To-Go is atrademark of Amersham Pharmacia Biotech Ltd.
(a)U.S. Pat. No. 6,027,945 and other patents pending.
(b)The PCR process is covered by patents issued and applicable in certain countries. Promega does notencourage or support the unauthorized or unlicensed use of the PCR process.

isolation from plant tissue andseeds. For more information,please see the Bioworks® methods available online at:www.promega.com/plantbio.

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Te c h n i c a l Q u e s t i o n s ?
E-mail Promega Technical Services at:techserv@promega.com