APPLICATION NOTE #201504

Genomic DNA Extraction from Plant Tissues on
the Fisher Scientific Bead Mill 24
Shari Garrett

Genetically modified crops (GM crops)
are food products such as fruits andvegetables that have had specific changes
introduced into their DNA. The DNAchanges are designed to impart newtraits into the crops such as resistance topathogens, delayed ripening or in the caseof hybrid soybeans produced by Pioneer
Hi-Bred International, higher levels ofoleic acid. Production of GM crops isa multistage process that ultimately
involves integration of the target gene
into the recipients’ genome. However,
uptake and integration of the gene into
the recipients genome does not always
guarantee that the gene will be expressed.
Furthermore, it must be confirmedthat the target gene is transferred at an
acceptable level to progeny.
In order to confirm gene expression,
genomic material (DNA/RNA) must firstbe extracted from the target sample.
Bead mill homogenizers are one of thenewest technologies for the disruptionof tough plant material and are routinely
used when multiple samples must beprocessed or when the target sample
is particularly robust such as seeds andtough plant material. The Bead Ruptor
Elite is capable of homogenizing samples
in volumes of 0.5 ml to 50 ml throughrapid oscillation of a tube containing
dense bead media. The bead media is
projected through the tube with sufficientenergy to impact and dissociate thesample. Herein, we demonstrate DNA
extraction from two common consumer
crops, spinach and okra using the Bead
Ruptor Elite.
The Bead Ruptor’s 24 extraction efficiency

and analyte integrity was evaluated.

Materials & Methods
Equipment•Omega Bio-tek - E.Z.N.A HP PlantDNA Mini Kit ( (Cat # D2485-00)•Bead Mill 24 (Cat No. 15340163)•Hard Tissue Homogenizing Mix(Cat No. 15340154)•Sigma - Antifoam Y-30 Emulsion(Cat #A5758)

Sample Preparation
Fresh spinach leaves and okra pods wereobtained from a local grocer and frozen
at 0°C overnight. Approximately 50 mgof spinach and okra was harvested andcut into 10 mm pieces and placed in a 2ml tube containing six 2.8 mm ceramicbeads (Cat # 15340154). 500 µl of lysis
solution provided from the E.Z.N.A HP
Plant DNA Mini Kit (Omega Bio-tek) and 5µl of antifoam were added and thesamples were homogenized on the Bead
Mill 24 at 4 m/s for 1 minute. After
processing, the lysates were transferred
to a clean 1.5 ml microcentrifuge tube
containing 10µl of 2-mercaptoethanol
and incubated at 65°C for 15 minutes.
The sample was inverted twice during
incubation. DNA extraction was carriedout per the manufacturer’s instructions
using the E.Z.N.A HP Plant DNA Mini Kit(Omega Bio-Tek). 1 µl of the eluted DNA
was quantified at 254 nm on a NanoDrop

Spectrophotometer (Thermo Fisher) todetermine DNA yields.
20 µl of each sample was then mixed with
10 µl of TBE/Urea sample buffer (BioRad Cat#161-0768). The DNA was thenseparated on a 1.2% TBE agarose gel at aconstant 60 V and stained with ethidiumbromide(Bio-Rad Cat#161-0433) for 30minutes. The gel was washed with DD
H20 for 10 minutes and visualized at 254
nm on a GelDoc EZSystem (Bio-Rad).

Results
Optimizing the sample disruption
process is a critical first step for nearly
every downstream genomic analysis.
Both analyte yields and integrity mustbe considered when selecting a sample
disruption method. Bead milling has
been demonstrated to be an effective
method of rapid sample dissociationfor extraction of nucleic acids from awide range of crop samples including
legumes, wheat and maize. In thisstudy, we evaluated the performance of
the Bead Mill 24 bead mill homogenizer
for the disruption of two common
crops, spinach and okra. Both plant
samples were disrupted through a one
minute cycle at 4 m/s. Visual inspection
confirmed that both samples werecompletely homogeneous after
processing on the Bead Mill 24.
Genomic DNA was then extracted andquantified by spectrophotometry (TableTissue Type

Mass (mg)

Average Concentration (ng /u L)

Spinach

45

3.25

Okra

50

9.83

Table 1 Starting sample quantity and DNA yields as determined by spectrophotometry.

Results (cont.)1). DNA yields were 3.25 ng/µl and 9.83 ng/µl for the spinach and okra samples respectively.
The DNA was then analyzed by gel electrophoresis to verify the quality of the extracted DNA. The gel image in figure 1 indicatesthat the extracted DNA was of good quality with relatively no shearing observed as demonstrated by the strong high molecularbands (Figure 1).

Figure 1 DNA agarose gel electrophoresis of pant lysates: Lane 1: 2.5kb ladder, Lane 2-3:
Spinach and Lane 5-6: OkraConclusion
The Bead Mill 24 is capable of disrupting both soft and fibrous plant tissue samples as demonstrated by the homogenization of
spinach and okra samples in one minute. The bead mill disruption process was demonstrated to be sufficient for extraction ofgenomic DNA in quantities sufficient for analysis by 1D gel electrophoresis and traditional staining and visualization procedures.
DNA integrity was maintained as well.

Bead Mill 24: (Cat No. 15340163)