Power Fecal™ DNA Isolation Kit: Instruction Manual
Power Fecal™ DNA Isolation Kit: Instruction Manual
Power Fecal™ DNA Isolation Kit: Instruction Manual
Quantity
50 Preps
Instruction Manual
Inhibitor Removal Technology (IRT) is a registered trademark of MO BIO Laboratories, Inc. and is covered by the following
patents USA US 7,459,548 B2, Australia 2005323451 and India 246946.
Please recycle
Version: 11022012
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
Table of Contents
Introduction .................................................................................................................................... 3
Protocol Overview........................................................................................................................... 3
Flow Chart................................................................................................................................... 4
Equipment Required ...................................................................................................................... 5
Kit Contents & Storage .................................................................................................................. 5
Precautions & Warnings ................................................................................................................ 5
Protocols:
Experienced User Protocol ................................................................................................ 6
Detailed Protocol (Describes what is happening at each step) ......................................... 8
Vacuum Manifold Protocol .. 11
Hints & Troubleshooting Guide ..................................................................................................... 13
Contact Information ....................................................................................................................... 15
Products recommended for you ................................................................................................. 16
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
Introduction
The PowerFecal DNA Isolation Kit is designed for fast and easy purification of both microbial and host
genomic DNA from stool and feces. Based on the MO BIO PowerSoil DNA Isolation Kit, the
PowerFecal DNA Isolation Kit uses the same patented Inhibitor Removal Technology (IRT) for stool
that has worked so well for soil. IRT is very effective at removing inhibitory substances commonly found
in stool such as polysaccharides, heme compounds and bile salts. The result is high purity DNA that is
ready to use in the most demanding downstream applications.
Protocol Overview
The recommended starting sample is 0.25 grams of stool or biosolid. Each sample is homogenized in a
2 ml bead beating tube containing garnet beads. Cell lysis of host cells as well as microbial cells is
facilitated by both mechanical collisions between beads and chemical disruption of cell membranes,
ensuring efficient extraction from even the toughest of microorganisms. Patented Inhibitor Removal
Technology (IRT) is then used to remove common substances in fecal samples that interfere with PCR.
Total genomic DNA is captured on a silica spin column. DNA is then washed and eluted so that it is
ready for PCR analysis and other downstream applications including qPCR and next generation
sequencing analysis.
Catalog No.
12988-10
12955-4
12955-12
13113-50
13116-50
13118-50
11991
11992
11992-10
11992-20
13156
13157
Quantity
10 preps
4 x 96 preps
12 x 96 preps
50 tubes
50 tubes
50 tubes
1 manifold
1 unit + 20 adapters
10 adapters
20 adapters
1 unit
1 unit
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
Equipment Required
Microcentrifuge (13,000 x g)
Pipettors (60 l - 750 l)
Vortex-Genie 2 Vortex (MO BIO Catalog# 13111-V or 13111-V-220)
Vortex Adapter (MO BIO Catalog# 13000-V1)
Kit Contents
Component
Dry Bead Tubes
Bead Solution
Solution C1
Solution C2
Solution C3
Solution C4
Solution C5
Solution C6
Spin Filters (units in 2 ml tubes)
2 ml Collection Tubes
Kit Storage
Kit reagents and components should be stored at room temperature (15-30C).
Precautions
Please wear gloves when using this product. Avoid all skin contact with kit reagents. In case of contact,
wash thoroughly with water. Do not ingest. See Material Safety Data Sheets for emergency procedures
in case of accidental ingestion or contact. All MSDS information is available upon request (760-9299911) or at www.mobio.com on the product page. Reagents labeled flammable should be kept away
from open flames and sparks.
WARNING: Solution C5 contains ethanol. It is flammable. Do not use bleach to clean the inside of the
PowerVac Manifold or to rinse the PowerVac Mini Spin Filter Adapters when attached to the
manifold.
IMPORTANT NOTE FOR USE: Shake to mix Solution C4 before use.
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
spin filters on to any vacuum manifold with Luer lock fittings. Please read Vacuum Protocol using
the PowerVac Manifold on page 11.
17. Add 500 l of Solution C5 and centrifuge for 1 minute at 13,000 x g.
18. Discard the flow through.
19. Centrifuge again for 1 minute at 13,000 x g.
20. Carefully place the Spin Filter in a clean 2 ml Collection Tube (provided). Avoid splashing any of
Solution C5 onto the Spin Filter.
21. Add 100 l of Solution C6 to the center of the white filter membrane. Alternatively, sterile DNA-Free
PCR Grade Water or TE buffer may be used for elution from the silica Spin Filter membrane at this
step (MO BIO Catalog# 17000-10).
Note: Eluting with 100 l of Solution C6 will maximize DNA yield. For more concentrated
DNA, a minimum of 50 l of Solution C6 can be used. Do not use less than 50 l of
Solution C6.
22. Centrifuge at 13,000 x g for 1 minute and discard the Spin Filter basket.
The DNA in the tube is now ready for any downstream application. No further steps are required.
We recommend storing DNA frozen (-20 to -80C). Solution C6 contains no EDTA. To concentrate
the DNA see the Hints & Troubleshooting Guide.
Thank you for choosing the PowerFecal DNA Isolation Kit.
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
3. Check Solution C1. If Solution C1 has precipitated, heat solution to 60C until dissolved before use.
Whats happening: Solution C1 contains SDS. If it gets cold, it will form a white precipitate in the bottle.
Heating to 60C will dissolve the SDS while not harming the SDS or the other disruption agents. Solution C1
can be used while it is still warm.
6. Secure the bead tubes horizontally using the MO BIO Vortex Adapter tube holder for the vortex (MO
BIO Catalog# 13000-V1) or secure tubes horizontally on a flat-bed vortex pad with tape. Vortex at
maximum speed for 10 minutes.
Whats happening: By shaking the beads in the presence of disruption agents, collision of the beads with
microbial cells will cause the cells to break open. The MO BIO Vortex Adapter is a simple platform to keep the
bead tubes tightly attached to the vortex during shaking. While one can attach the tubes with tape, the tape can
become loose so that the tubes shake unevenly and inefficiently resulting in inconsistent results or lower yields.
9. Add 250 l of Solution C2 and vortex briefly to mix. Incubate at 4C for 5 minutes.
Whats happening: Solution C2 is patented Inhibitor Removal Technology (IRT). It contains a reagent to
precipitate non-DNA organic and inorganic material including polysaccharides, cell debris, and proteins. It is
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit
downstream DNA applications.
12. Add 200 l of Solution C3 and vortex briefly. Incubate at 4C for 5 minutes.
13. Centrifuge the tubes at 13,000 x g for 1 minute.
14. Avoiding the pellet, transfer the supernatant to a clean 2 ml Collection Tube (provided).
Do not transfer more than 750 l at this step.
Whats happening: The pellet at this point contains additional non-DNA organic and inorganic material. For the
best DNA yield and quality avoid transferring any of the pellet. It is important that no more than 750 l be
transferred to the next step to ensure proper binding of the DNA to the silica spin column.
15. Shake to mix Solution C4 before use. Add 1200 l of Solution C4 to the supernatant and vortex for
5 seconds.
Whats happening: Solution C4 is a high concentration salt solution. Since DNA binds tightly to silica at high
salt concentrations, this will adjust the DNA solution salt concentrations to allow binding of DNA, but not nonDNA organic and inorganic material that may still be present at low levels, to the Spin Filters.
16. Load 650 l of supernatant onto a Spin Filter and centrifuge at 13,000 x g for 1 minute. Discard the
flow through and repeat until all the supernatant has been loaded onto the Spin Filter.
Note: A total of three loads for each sample processed are required.
High Throughput Option: Step 16 can become tedious when many samples need to be processed.
For this reason, MO BIO has developed a vacuum protocol. It does require the purchase of our
aluminum Spin Filter Adapters (MO BIO Catalog# 11992-10) which will allow you to fit our flat bottom
spin filters on to any vacuum manifold with Luer lock fittings. Please read Vacuum Protocol using
the PowerVac Manifold on page 11.
Whats happening: DNA is selectively bound to the silica membrane in the Spin Filter device in the high salt
solution. Contaminants pass through the filter membrane, leaving only DNA bound to the membrane.
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
20. Carefully place the Spin Filter in a clean 2 ml Collection Tube (provided). Avoid splashing any of
Solution C5 onto the Spin Filter.
21. Add 100 l of Solution C6 to the center of the white filter membrane. Alternatively, sterile DNA-Free
PCR Grade Water or TE buffer may be used for elution from the silica Spin Filter membrane at this
step (MO BIO Catalog# 17000-10).
Note: Eluting with 100 l of Solution C6 will maximize DNA yield. For more concentrated
DNA, a minimum of 50 l of Solution C6 can be used. Do not use less than 50 l of
Solution C6.
Whats happening: Placing Solution C6 (sterile elution buffer) in the center of the small white membrane will
make sure the entire membrane is wetted. This will result in more efficient and complete release of the DNA
from the silica Spin Filter membrane. As Solution C6 (elution buffer) passes through the silica membrane, DNA
that was bound in the presence of high salt is selectively released by Solution C6 (10 mM Tris) which lacks salt.
22. Centrifuge at 13,000 x g for 1 minute and discard the Spin Filter basket.
The DNA in the tube is now ready for any downstream application. No further steps are required.
We recommend storing DNA frozen (-20 to -80C). Solution C6 contains no EDTA. To concentrate
the DNA see the Hints & Troubleshooting Guide.
Thank you for choosing the PowerFecal DNA Isolation Kit.
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
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Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
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10. Discard the Spin Filter column. The DNA in the tube is now ready for any downstream application.
No further steps are required.
We recommend storing DNA frozen (-20 to -80C). Solution C6 contains no EDTA. To concentrate
the DNA see the Hints & Troubleshooting Guide.
Thank you for choosing the PowerFecal DNA Isolation Kit.
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
12
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
13
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
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Contact Information
Technical Support:
Phone MO BIO Laboratories, Inc. Toll Free 800-606-6246, or 760-929-9911
Email: [email protected]
Fax: 760-929-0109
Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA 92010
Ordering Information:
Direct: Phone MO BIO Laboratories, Inc. Toll Free 800-606-6246, or 760-929-9911
Email: [email protected]
Fax: 760-929-0109
Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA 92010
For the distributor nearest you, visit our web site at www.mobio.com/distributors
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
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For a complete list of products available from MO BIO Laboratories, Inc., visit www.mobio.com
Description
Catalog No.
Quantity
26000-50
50 preps
27500-4-EP
4 x 96 preps
13111-V
1 unit (120V)
13111-V-220
1 unit (220V)
13000-V1-24
12500-50
12500-100
12500-250
50 preps
100 preps
250 preps
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
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