Standard Operating Procedure
Title: Rapid Isolation of Genomic DNA from Soybean Roots using FastDNA® SPIN
Kits
Department: Agronomy
Created by: Catherine Swoboda
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr. Palle Pedersen
Lab Supervisor: Maria Hartt
Date approved: 12 June 2008
Procedure Overview: The FastDNA® SPIN Kit isolates genomic DNA from various
sources. It is designed for use with FastPrep® Instruments. These instruments can
homogenize soybean root samples through impaction with lysing matrix particles.
After lysis, the samples are centrifuged to pellet debris and lysing matrix. DNA is
purified from the supernatant using a silica-based GENECLEAN® procedure using
SPIN filters. The eluted DNA will then be analyzed using Quantitative PCR. The
Quantitative PCR analysis will be performed in the Department of Plant Pathology at
the University of Wisconsin at Madison (contact Dr. Paul Esker:
pde@plantpath.wisc.edu).
Equipment and reagents necessary:
FastDNA® SPIN Kit Components
Lysing Matrix A
¼” Ceramic Spheres
Binding Matrix
Concentrated SEWS-M
DES
CLS-VF
PPS
CLS-TC (not used)
CLS-Y (not used)
SPIN Modules
Catch Tubes
User Supplied Materials
100 x 2.0 ml tubes
100 spheres
66 ml
12 ml
25 ml
90 ml
25 ml
110 ml
110 ml
100 each
100 each
100ml graduated cylinder
Ethanol, 100%, 100 ml per 100 samples
Soybean root samples
Balance
Weigh boat
Spatula
Pipette and tips, 100, 200, 500 & 1000µl
Microcentrifuge able to spin 2.0 ml tubes
Microcentrifuge tubes (2.0 and 1.5 ml)
Microcentrifuge tube rack
Rotator or low-speed vortex
Tongs
Heat block or water bath, 55C
FastPrep® Instrument
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Solution preparation:
SEWS-M Wash Solution: Add 100 ml of 100% tax-free ethanol to the bottle
containing 12 ml of Concentrated SEWS-M Wash Solution. Securely close the bottle
in order to prevent evaporation. Label the bottle with the date the ethanol was
added. Store Wash Solution at room temperature.
Procedure:
1. Add between 100 – 200 mg of soybean root sample to Lysing Matrix A tube (tissue
can be fresh, frozen, dried etc.).
2. Add 800µl CLS-VF (Cell Lysis Solution) and 200µl PPS (Protein Precipitation
Solution) to tube. All pipette tips should be disposed of in a broken glass
receptacle.
3. Homogenize in the FastPrep® Instrument at a speed setting of 6.0 for 40 seconds.
4. Centrifuge at 14,000 x g for 10 minutes to pellet debris.
5. Transfer supernatant (~700 µl) to a 2.0 ml microcentrifuge tube. Add an equal
volume of Binding Matrix. Invert tube to thoroughly mix contents.
It is important to note that tubes with conical bottoms do not work well for mixing the
entire volume. A 2.0 ml microcentrifuge tube works well as it allows for the entire
volume to be completely mixed.
6. Incubate with gentle agitation at room temperature for 5 minutes on a rotator. A lowspeed vortex can be used in this step, but the DNA cannot be sheared.
7. Remove tube from rotator and pipette approximately 700 µl (half) of the suspension
into a SPINTM Filter/Catch Tube assembly; centrifuge at 14,000 x g for 1 minute.
Empty the contents of the Catch Tube into an appropriately tagged waste container;
return SPINTM Filter to Catch Tube. Add the remaining suspension to the SPINTM
Filter and centrifuge as before. Empty the Catch Tube into waste container; return
SPINTM Filter to Catch Tube.
8. Add 500 µl of the prepared SEWS-M Wash Solution to SPINTM Filter then gently
resuspend the pellet using the force of the liquid from the pipette tip. It is important
that the ethanol has been added to the Concentrated SEWS-M.
9. Centrifuge at 14,000 x g for 1 minute. Empty the contents of the Catch Tube into
waste container; return SPINTM Filter to Catch Tube.
10. Do not add more liquid. Centrifuge a second time at 14,000 x g for 2 minutes then
replace the Catch Tube with a new, clean tube. Empty contents of first Catch Tube
into waste container; discard Tube.
11. To elute the DNA, add 100 µl of DES to SPIN filter and gently pipette solution up and
down to resuspend the residual Binding Matrix (added in step 5) above the SPIN
filter. Incubate sample by using tongs to place tube in a 55ºC heat block or water
bath for 5 minutes. Use tongs to remove tubes.
12. Centrifuge at 14,000 x g for 1 minute in order to bring eluted DNA into the clean
catch tube. Discard the SPIN filter. At this point, DNA is ready for downstream
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applications. Store tubes in an appropriately labeled, enclosed container at -20ºC for
extended periods or at 4ºC until use.
13. Pour any unwanted solutions into waste container. Dispose of dried pellets in trash.
14. Clean work area.
Personal Protective Equipment / Engineering Controls:
Nitrile gloves
Safety glasses
Face shield
Dust mask
Latex gloves
Splash goggles
Lab coat
Fume hood
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
Handling & Storage Precautions:
Binding Matrix (Silica Gel Suspension in Water): Store at room temperature in a dry location.
CLS-VF: Use dustless method systems (water or vacuum) for handling, storage and clean-up
so that exposure does not exceed TLV established by OSHA 1910.1000.
DNase Free Water (DES): Store DES in an appropriately labeled, tightly sealed container.
Store in a cool, dry place and keep away from incompatible substances when using or storing.
Ethanol Wash Solution (SEWS-M): Solution is an irritant. Wear protective clothing and rubber
gloves. Flush small quantities down the sink with water to dispose.
Potassium Acetate, Acetic Acid (PPS): Store in an appropriately labeled, tightly closed
container. Store in a cool, dry location away from ignition sources and oxidizers.
Waste Disposal Procedures:
Unless EH&S specifically instructs otherwise, all chemical/reagent waste (including excess
solutions) must be placed in an appropriately labeled hazardous waste container for EH&S
disposal. Compatible substances may be combined into one waste container.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Binding Matrix: Contain spill; place in an appropriately labeled hazardous waste container for
EH&S disposal.
CLS-VF: Clean up by recommended dustless methods (water or vacuum). Do not exceed
OSHA TLV Standard (1910.1000 Table Z-3); place in an appropriately labeled hazardous
waste container for EH&S disposal.
DES: Dam larger spills (of solution) with inert materials to prevent spread. To dispose of
wastes, place in an appropriately labeled hazardous waste container for EH&S disposal.
PPS: Dike spill, take up with absorbent and containerize for proper disposal. To dispose,
place in an appropriately labeled hazardous waste container for EH&S disposal.
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SEWS-M: Wear protective clothing and rubber gloves when handling and in the case of a spill.
To dispose, place in an appropriately labeled hazardous waste container for EH&S disposal.
Health & Safety Summary for Required Reagents:
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Binding matrix contains guanidine thiocyanate an organic compound that can ignite. Avoid
strong oxidants and string acids.
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Overexposure of free silica dust from CLS-VF may cause delayed lung injury.
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Overexposure to PPS will cause headache, dizziness and muscle weakness.
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Overexposure to SEWS-M causes skin and eye irritation and irritation to mucus
membranes and respiratory tract.
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Guanidine
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Incompatibilities
Avoid strong oxidants,
string acids.
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 The above summary consists of guidelines for proper handling & disposal of
chemicals used in this procedure. You must read and understand the
contents of the entire MSDS(s) before starting this procedure.
References:
DNA was isolated from soybean root samples using the FastDNA® SPIN Kit and the
FastPrep® Instrument (MP Biomedicals, Santa Ana, CA).
2008. Instruction Manual FastDNA® SPIN Kit. Rapid Isolation of Genomic DNA from
Plant and Animal Tissue, Bacteria, Yeast, Algae and Fungi Using the FastPrep®
System. MP Biomedicals, Santa Ana, CA.
*NOTE: Protocol presented above, taken from Instruction Manual FastDNA® SPIN Kit.
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