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Protocol: High Throughput Isolation of Plant DNA using the 96 Well Synergy™ Plant DNA Extraction Kit

Introduction

The 96 Well Synergy™ Plant DNA Extraction Kit is a bead beating based kit, allowing for high throughput sample processing. Impurities are captured on the Synergy™ grinding matrix and DNA in the lysate is further purified using filter plates. Unlike most commercially-available kits, the Synergy™ kit includes pre-filled deep well plates for sample homogenization. Individual strips of tubes can be removed from the plate for partial plate homogenization. Plant samples are loaded into each well and bead beated in a plate homogenizer until samples are fully homogenized. Mechanical disruption results in less sample variation than manual disruption using a mortar and pestle.

Materials and Methods

To validate the 96 Well Synergy™ Plant DNA Extraction Kit, various plant samples were tested using the Synergy™ chemistry. Samples  included Arabidopsis, corn, duckweed, rye, sorghum, tobacco and wheat.

To start, 40 mg of plant samples were added to each well of the 96 Well Synergy™ Homogenization Plate, followed by the addition of 350 µl of Plant Homogenization Buffer. Wells were sealed with strip caps, and the Well Support Mat was placed underneath the loaded plate. Samples were bead beated for 13 minutes at 1,500 rpm in a high velocity plate homogenizer until the wells lacked foam. The plate was centrifuged for 10 minutes at 4,000 rpm. Next, 120 µl of supernatant from each well was transferred to a PVDF Filter Plate, with a Collection Plate placed underneath, and centrifuged for 10 minutes at 4,000 rpm. RNase A Solution (5 µl) was added to the filtrates and incubated at room temperature for 15 minutes.  Isopropanol (98 µl) was added to the solutions, thoroughly mixed by pipetting, and then incubated at -20°C for 15 minutes. A Collection Plate was placed under the Glass Fiber Filter Plate. Solutions were transferred to the Glass Fiber Filter Plate, and the plates were centrifuged for 10 minutes at 4,000 rpm. Flow through from the Collection Plate was discarded, and the plate was placed back under Glass Fiber Filter Plate. DNA bound to the filter plate was washed with 250 µl of ice-cold 70% ethanol in each well. The plates were centrifuged for 10 minutes at 4,000 rpm. Flow through from the Collection Plate was discarded, and the plate was placed back under Glass Fiber Filter Plate. Samples were washed once more with ethanol and centrifuged. The Elution Plate was placed underneath the Glass Fiber Filter Plate, and 50 µl of Molecular Biology Grade Water was added to each well and centrifuged for 11 minutes at 4,000 rpm, eluting the DNA. Samples were then analyzed on a spectrophotometer. Click here for a visual representation.

Results and Discussion

Based on the results, the 96 Well Synergy™ Plant DNA Extraction Kit effectively isolated DNA from all tested samples (Table 1).

Table 1: DNA yields and purities from various plant samples.

Sample

Concentration

260/280 Ratio

260/230 Ratio

Arabidopsis

218.83 ng/µl

1.98

2.06

Corn

174.70 ng/µl

1.94

2.15

Duckweed

101.44 ng/µl

1.91

2.10

Rye

157.44 ng/µl

1.83

2.25

Sorghum

110.36 ng/µl

1.85

1.97

Tobacco

229.43 ng/µl

1.91

2.18

Wheat

94.41 ng/µl

1.83

2.08

DNA concentrations ranged from 94.41 ng/µl to 229.43 ng/µl. The 260/280 ratios indicated pure DNA, as averages ranged from 1.83 to 1.98. The ratios greater than 1.9 may be explained by residual RNA in the mature plant samples. All 260/230 ratios were approximately 2.0, suggesting there were no carbohydrates, polyphenols or EDTA in the samples.

It is important to recognize that all plant samples are not the same. If mature or frozen plant samples are used and ratios are not optimal, the protocol can be adjusted to use a smaller starting sample mass and buffer volume. While DNA concentrations will decrease, the purity of the DNA will increase, which is critical for downstream applications, including DNA sequencing. If multiple plant species are placed into one 96 Well Homogenization Plate, it is crucial to achieve thorough homogenization for all plant types. Some plants will homogenize in 10 minutes, while others may take up to 15 minutes.

To summarize, the Synergy™ chemistry, modified for the 96 well format, successfully isolated high concentrations of high quality DNA from various plant samples in a high throughput and cost effective manner.