Fungal DNA Isolation with PowerPlant Pro DNA Isolation Kit (MO BIO)
Nimalka M Weerasuriya
Abstract
This is a slightly modified MOBIO PowerPlant Pro DNA Isolation Kit for Bennett Plant Pathology.
Technical information:
Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com
Introduction
The PowerPlant® Pro DNA Isolation Kit is designed for fast and easy purification of total cellular DNA
from plant cells, tissues and seeds. The bead beating technology used in this kit replaces cumbersome
DNA isolation procedures such as CTAB, phenol, or chloroform extraction for recovery of high quality
DNA from the toughest sample types, including strawberry leaf, cotton leaf, cotton seeds, and pine
needles. The PowerPlant® Pro DNA Isolation Kit utilizes our patented Inhibitor Removal Technology®
(IRT) for removal of PCR inhibitors from plant extracts during the isolation process, resulting in DNA that
is ready to use in any downstream applications including PCR, qPCR and sequencing
Before start
Mechanical Lysis Options
The PowerPlant® Pro DNA Isolation Kit may be used with a vortex or high velocity bead beater, such as
the PowerLyzer™ 24 homogenizer. The PowerLyzer™ 24 is suitable for fast homogenization of plant
materials including stems, roots, seeds or difficult leaf tissue without the need of liquid nitrogen grinding
The PowerLyzer™ 24 is a highly efficient bead beating system that allows for optimal DNA extraction
from a variety of plant tissues. The instrument’s velocity and proprietary motion combine to provide the
fastest homogenization time possible, minimizing the time spent processing samples. The
programmable display allows for hands-free, walk-away extraction with up to ten cycles of bead beating
for as long as 5 minutes per cycle. This kit provides Bead Tubes prefilled with 2.38 mm stainless steel
beads for homogenizing plant tissue for optimal DNA isolation. Alternative pre-filled bead tube options
are available for additional applications. Please contact technical service (technical@mobio.com) for
details.
For isolation of DNA using this kit with the FastPrep® or Precellys®, the following conversion chart will
help you to adapt your current protocol. However, due to the highly efficient motion of beads in the
PowerLyzer™ 24, we have found that less cycle numbers are required to generate the same effect. You
may want to perform extractions on the PowerLyzer™ 24 at the equivalent speed and number of cycles
as your current instrument and compare it to less time or lower speed to determine which settings give
the best results.
- The Bennett lab uses the Thermo Savant FastPrep FP120.
Steps
Preamble
Begin this extraction protocol after growing Pythium or other fungal isolates on 1/2-strength PDA or full-strength PDA (recommended) for 5-7 days.
Detailed Protocol
Add 450µL of Solution PD1 into each 2 ml PowerPlant Bead Tubes provided.
To each tube scrape 0.1mL hyphae into microcentrifuge tube using pipette tip, scalpel or sterile toothpick. Avoid scraping agar.
- Pipette tips or toothpicks can be useful when initially breaking down the hyphal mat. Scrape the side of the tip or toothpick against the inside of the tube to create an opaque solution.
Check Solution PD2 for precipitates, if precipitated, warm at 37°C - 55°C until dissolved. Add 50µLof Solution PD2 .
Add 3µL of RNase A Solution to the PowerPlant® Bead Tube and vortex briefly to mix.
Homogenize using one of the following methods:
2. PowerLyzer™ 24 Homogenizer : Properly identify each PowerPlant Bead Tube on both the cap and on the side.
Place Bead Tubes into the Tube Holder of the PowerLyzer™ 24. The PowerPlant Bead Tubes must be balanced (evenly spaced) on the Tube Holder. Homogenize the tissue for 1 cycle at the chosen speed depending on your sample type for 2 minutes.
3. Other Homogenizers: The Bennett Lab uses a FastPrep FP120. You may want to perform extractions at the equivalent speed and number of cycles as your current instrument and compare it to less time or lower speed to determine which settings give the best results.
Fungal mycelium may be best homogenized on the FP120 at ``
| A | B | C | D | E |
|---|---|---|---|---|
| Plant Tissue Type | PowerLyzer Speed | FastPrep Speed (24 m/s) | No. of Cycles | Time/Cycle |
| Soft leaf tissues | 2000 RPM | NA | 1 | 2 min |
| Fibrous leaf tissue | 2200 RPM | NA | 1 | 2 min |
| Stems | 2200 RPM | 1 | 2 min | |
| Roots | 2500 RPM | 4 | 1 | 2 min |
| Pine needles | 2600 RPM | 4 | 1 | 2 min |
| Seeds | 2800 RPM | 4.5 | 1 | 2 min |
| Fungal mycelium | 3700 RPM | 6 | 1 | 30 sec |
Suggested homogenization times for recommended and available equipment.
Centrifuge Bead Tubes at 13000x g (RCF).
Transfer the supernatant to a clean 2mL Collection Tube (provided).
Add 175µL of Solution PD3 . Vortex 0h 0m 5s. Place on ice or refrigerated rack at 4°C for 0h 5m 0s.
Centrifuge the Collection Tube at 13.000x g (RCF).
Avoiding the pellet, transfer up to 600µL of supernatant to a clean 2 ml Collection Tube (provided).
Add 600µL of Solution PD4 and 600µL of Solution PD6 . Vortex to mix for 0h 0m 5s.
Load approximately 600µL of lysate onto the Spin Filter and centrifuge at 10.000x g (RCF). 1. Discard the flow through, place the Spin Filter back into the Collection Tube and add another 600µL of lysate and centrifuge at 10.000x g (RCF).
- Discard the flow-through and repeat a third time until all of the lysate has been passed through the Spin Filter.
- Discard the flow-through and place the Spin Filter back into the Collection Tube.
Note
What’s happening: In the presence of Solution PD4 & Solution PD6, DNA will bind to the spin filter. Centrifugation of the combined lysate through the spin filter allows the DNA to bind the filter membrane while allowing unwanted salt and impurities to pass through the membrane.
Add 500µL of Solution PD5 to the Spin Filter column. Centrifuge for 10.000x g (RCF). Discard the flow through. Place the Spin Filter back into the same Collection Tube.
Add 500µL of Solution PD6 to the Spin Filter column. Centrifuge for 10.000x g (RCF). Discard the flow through. Place the Spin Filter back into the same Collection Tube.
Centrifuge for 16.000x g (RCF) to remove residual Solution PD6 .
Carefully place the Spin Filter into a new clean 2 ml Collection Tube (provided). This is your final collection tube, label accordingly. Avoid splashing any Solution PD6 onto the Spin Filter .
Add 50-100µL of Solution PD7 (10 mM Tris, pH 8.0) to the center of the white filter membrane and incubate for 0h 2m 0s at Room temperature.
Centrifuge 10.000x g(RCF).
Centrifuge 10.000x g (RCF).
Discard the Spin Filter . DNA in the tube is now ready to use. No further steps are required.
We recommend storing DNA frozen -20°C. Solution PD7 contains no EDTA
Nanodrop
Use the elution buffer (Solution PD7) as your blank in Nanodrop.
Nanodrop Lite (Shared Equipment Lab)
Please enter the sample names and DNA concentrations and A260/280 ratios as a note.
