Application Note

Rapid, High Performance and Cost-Effective Plant DNA Extractions

Kiranmai Durvasula1, Jeff Roeder1, Travis Butts1
1
Omega Bio-tek, Inc.
Spin columns Magnetic beads

Introduction

The incredible diversity of the plant kingdom comes with a wide Lyse sample
range of DNA extraction challenges. These challenges include
extensive variations in plant structures and in their chemical
composition of polysaccharides, polyphenolic compounds, and
humic substances, all of which can interfere with extraction Precipitate
efficiency and/or inhibit downstream applications. In this polysaccharides Bind DNA
context, Omega Bio-tek has developed a highly versatile DS line
of plant DNA extraction kits encompassing both manual and
automated extraction realms. The kits utilize the well-established
properties of the cationic detergent, cetyltrimethylammonium
Magnetize &
bromide (CTAB), in conjunction with our proprietary silica Bind DNA remove
column chemistries (manual) and magnetic bead (automated) supernatant
to extract high-quality DNA from a variety of plants.

Materials and Methods

Wash
To demonstrate Omega Bio-tek’s plant extraction capabilities,
a variety of plant samples were subjected to automated and
manual extraction performance testing. The manual extractions
were performed with Omega Bio-tek’s E.Z.N.A.® Plant DNA DS
(D2411) spin column-based kit alongside a similar product from Dry
a leading competitor (Company Q). The automated extractions
were performed using Omega Bio-tek’s Mag-Bind® Plant DNA
DS 96 Kit (M1130) ported onto an open-ended, programmable,
robotic liquid handler (Qiagen’s BioSprint® 96). Plant specimens
used for the experiments were either acquired commercially or
grown at Omega Bio-tek. All the extractions for each method Elute
tested were performed in triplicate following manufacturer’s
recommended protocols. Each sample consisted of
approximately 40-50 mg (wet weight) of plant leaf material and
was disrupted at 1,500 rpm.
Figure 1. The E.Z.N.A. Plant DNA DS Kit (D2411) uses silica spin columns and is
the manual process shown above. The Mag-Bind Plant DNA DS 96 Kit (M1130)
The workflows for both manual and automated extractions uses magnetic beads and is the automated process shown above. Omega Bio-
are shown in Figure 1. Resulting purified DNA was quantified tek’s recommendation is to use magnetic bead-based protocols for automated
via Promega’s QuantiFluor® dsDNA system and normalized processing.
per mg of input plant material. The quality of the purified DNA
extracted from corn samples using silica spin column-based kits kit over Company Q’s kit. Also, the ΔCt values with the Omega kit
from Omega Bio-tek (D2411) and Company Q was assessed by were closer to 3.3 that of Company Q (~2.94 vs. ~1.87 difference
real-time PCR. Briefly, undiluted, 10-fold and 100-fold dilutions between 10-fold and undiluted) suggesting less qPCR inhibition
of purified DNA were isolated and amplified using Agilent’s in spite of more template DNA that seems to have been present.
Brilliant III 2X SYBR® mix and corn-specific primers following a The results from the qPCR indicate that Omega Bio-tek’s kit was
standard amplification protocol on the ABI 7900. not only able to extract more DNA, but was also able to remove
potential plant-related inhibitors that can hamper various
Results downstream applications.

The DNA yields obtained following manual protocols using Twenty-three of the top agricultural and biofuel crops were
Omega Bio-tek’s kit were significantly higher than the leading subjected to automated extraction using Omega Bio-tek’s
competitor’s kit on all 5 different plant samples tested (Figure M1130 kit on Qiagen’s BioSprint® 96 and compared to the
2). Table 1 shows the average Ct values obtained on serial manual protocol of Company Q’s.
dilutions of purified DNA from corn using both kits. On average
across the dilutions, the Cts with the Omega extraction were The results shown in Table 2 reveal that the automated
lower by ~4.9 cycles compared to Company Q’s. The Ct values methods resulted in significantly higher recoveries of DNA
suggest that there is at least 16x more DNA using the Omega than the competitor’s manual method of 21 of the 23 plants

Innovations in Nucleic Acid Purification 1 Lit No. SL-0050

 Application Note

Table 2. DNA yield comparison from 23 different plant types. 50 mg leaf sample
150 Omega Bio-tek extracted per sample using Omega Bio-tek’s Mag-Bind Plant DNA DS 96 Kit
Company Q (M1130) and Company Q’s recommended protocols. The lysis and binding
buffers in the Mag-Bind Plant DNA DS 96 Kit and the E.Z.N.A. Plant DNA DS Kits
120 are identical. DNA concentration determined via fluorescence-based nucleic
acid quantification. Total yield was divided by total tissue amount to show ng of
DNA per mg of leaf tissue.
90
Mag-Bind Plant DNA
ng/mg

Plant Type Company Q

DS 96 Kit (M1130)
60
Hay 27.2 104.6
Tobacco 12.3 19.4
30
Peanut 6.3 52.9
Sunflower 41.8 89.1
0
Orange 4.6 31.2
a

co
ol

tto

ea
Co

Switchgrass 21.9 7.9

ac
n

yb
Co
Ca

b
To
So

Figure 2. 40-50 mg of respective fresh leaf tissue was extracted in triplicate Pepper 6.9 111.0
according to the E.Z.N.A. Plant DNA DS Kit protocol and Company Q’s
recommended protocols and eluted in 100 µL. DNA analyzed with fluorescent Sugarcane 10.5 93.1
DNA-based quantification method. Total yield was divided by total tissue amount
Jatropha 7.5 19.0
to show ng of DNA per mg of leaf tissue.
Oats 18.4 270.0
tested. Because the extraction methods being tested are based Wheat 0.5 152.3
on different chemistries, real-time PCR for the beta-tubulin Barley 9.6 198.1
gene was performed on a subset of 100-fold dilutions of plant
Canola 3.4 59.0
extracts from both the methods. The differences in the resulting
real-time PCR cycle threshold values were consistent with the Tomato 2.6 120.2
amount of template measured to be present in each extract, or Apple 5.7 121.8
when adjusted to contain equivalent (1 ng) amounts of template Grape 1.9 212.4
(data not shown). These results indicate that matrix effects have
Alfalfa 17.9 85.2
not substantially influenced reported DNA recovery results.
Corn 4.0 29.8
Conclusions Sugar beet 20.2 34.0
Soybean 26.8 25.4
Omega Bio-tek’s spin column and magnetic bead-based
approaches for plant DNA extraction performed significantly Cotton 30.5 63.5
better than its competitor. Overall, automated methods were Sorghum 29.4 72.1
much faster and required much less hands-on time than either Potato 30.0 206.5
of the manual methods. Up to 96 samples if 50 mg wet tissue
(or 15 mg dry tissue) can be processed in parallel in less than
1 hour using Omega Bio-tek’s automated approach. Omega Product Information
Bio-tek has leveraged their plant extraction experience and
expertise to develop a robust solution for plant DNA extraction Description Product No. Preps
needs. Options are available for automated on open liquid
handling and magnetic processor platforms, such as the D2411-00 5
E.Z.N.A.® Plant DNA DS Kit
Hamilton Microlab® STAR™, Hamilton Firefly® NIMBUS 96, and D2411-01 50
the Thermo Fisher Scientific KingFisher® Flex. D1411-00 1 x 96
E-Z 96® Plant DNA DS Kit
D1411-01 4 x 96

M1130-00 1 x 96
Table 1. Real-time PCR with corn-specific primers was performed on the triplicates Mag-Bind® Plant DNA DS 96 Kit
of undiluted, 10-fold and 100-fold dilutions of DNA. DNA isolated using Omega M1130-01 4 x 96
Bio-tek’s E.Z.N.A. Plant DNA DS Kit and a comparable column-based kit from
Company Q. Omega Bio-tek’s kit not only has significantly higher yields, but also
less qPCR inhibition when compared to that of Company Q’s.

Extraction Average Ct ΔCt

Method for Corn 400 Pinnacle Way, Suite 450
1X 10X 100X (10X-1X) (100X-10X)
Norcross, GA 30071
Omega Bio-tek 2.388 26.324 29.978 2.935 3.654
www.omegabiotek.com
Company Q 28.703 30.573 35.038 1.870 4.465
info@omegabiotek.com
(800) 832-8896

Innovations in Nucleic Acid Purification 2 Lit No. SL-0050