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SOP# 009 Version Eff ective Date: 2Dec2019

Title: DNA Purification from Fecal Samples

Scope: This protocol is for the purification of genomic DNA
from fecal samples in OMNIgene GUT tubes
Responsibility: Research Personnel Karnes Lab

1. Background
The AllPrep PowerFecal DNA/RNA Kit is designed to purify microbial
DNA and RNA
simultaneously from the same stool sample, while separating the
DNA and RNA into separate
eluate fractions. Purified DNA has an A260/A280 ratio between 1.7-
1.9. DNA can be safely stored at -80 C.
NoteNOTE: RNA will typically have a higher 260/280 ratio due to the
higher ratio of Uracil compared to that of Thymine.

2. Safety
This SOP is Biosafety Level 2 (BSL-2) and 1) all laboratory personnel
have specific training in handling pathogenic agents; 2) all
procedures involving direct human sample contact require disposal
via biohazard waste. This procedure is to be done at a BSL-2
designated bench area. When working with chemicals, always wear
applicable personal protective equipment (PPE).

3. Specimen
3.1 Specimen Type
Fecal samples collected in OMNIgene GUT collection tube.
3.2 Minimum Specimen Volume
Minimum sample 0.1 - 0.2g of stool sample
3.3 Handling Conditions
3.3.1 Samples are received from Heidi Steiner at Banner
UMC or shipped back directly to the lab
3.3.2 Fecal samples may be stored at room temperature (15-
25 C) for up to 60 days

4. Materials and Equipment

4.1 Preparation and Processing
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4.1.1 AllPrep PowerFecal DNA/RNA Kit

4.1.2 Tissue Wipers
4.1.3 Water bath with shaker
4.1.4 100% Ethanol
4.1.5 80% Ethanol
4.1.6 15mL centrifuge tubes
4.1.7 Pipets, tips
4.1.8 Vortexer
4.1.9 Vortexer adapater
4.1.10 microcentrifuge
4.1.11 Ice bucket, ice
4.1.12 Dithiothreitol (DTT), 1 M
4.1.13 Disposable Gloves
4.1.14 Ice machine located on 2nd floor
4.2 Storage
4.2.1 NanoDrop spectrophotometer
4.2.1.1 Dr. Smith lab
4.2.2 DYMO LabelWriter 450
4.2.2.1 Computer Karneslab3
4.2.3 1.5mL micocentrifuge tubes
4.2.4 Cryogenic labels

5. Procedure
5.1 Pre-Purification
5.1.1 Preheat water bath to 55 C
5.1.2 Warm Solution PM1 to use at 55°C for 10 min.
NOTE: Use Solution PM1 is used while still warm.
5.1.3 Prepare Ice Bucket
5.1.4 Buffers AW1, AW2 and RPE are supplied as
concentrates. Add the appropriate amounts of ethanol
(96–100%) as indicated on the bottles (see Important
Notes, page 10). Mix well after adding ethanol.

NOTE: once per kit

5.1.5 Prepare 80% ethanol in water.

5.1.6 For each prep, place a RNeasy Mini Spin Column in a
Collection Tube (2 mlmL).

1.1 Purification
1.1.1 Lysis
1.1.1.1 Vortex the samples in the OMNIgene GUT tube
for 10 seconds.
1.1.1.2 Pipette 125uL of sample from OMNI gene GUT
tube into a Microbial Lysis Tube
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1.1.1.3 Add 650 μl Solution PM1 and 25 μl DTT to the

Microbial Llysis Ttube and . tTightly cap the lid.
Note:NOTE: Prior to use, Solution PM1 must be
warmed at 55°C for 10 min to dissolve precipitates
(see Notes before starting, above). Use Solution PM1
while still warm.
1.1.1.4 Vortex at <speed> for MAX 5 minutes to lLyse
bacterial cells. with vortexer and adapter at maximum
speed for 5 minutes.
NOTE: Use vortex adapter so you can leave the sample.

1.1.1.5 Centrifuge at 18,000 x g for 1 min at room

temperature.
1.1.1.6 Transfer supernatant to a clean 1.5mL
collection tube
Note:NOTE: Eexpected a total volume of about 400-
500 μl supernatant at this step.
1.1.1.7 Add 150 μlL Solution IRS to the transferred
supernatant, close the lid gently and vortex briefly
to mix.
1.1.1.8 Incubate at 2–8°C for 5 min.
1.1.1.9 Centrifuge at 13,000 x g for 1 min at room
temperature.
1.1.1.9.1Avoiding the pellet, transfer 300 μl of the
supernatant to a clean 2 mLl Collection Tube.
1.1.1.9.2Note: Expect a total volume of ~400–500 μl
supernatant at this step.
1.1.1.9.3Optional: Users who want to maximize nucleic
acid yield may transfer up to 450 μl
supernatant at this step. However, volumes
greater than 300 μl will require additional
pipetting.
1.1.1.10 Shake to mix Solution C4 before use. Add 400 μl
Solution C4 to the transferred supernatant and mix
well by pipetting. Do not centrifuge.

NOTE: Shake to mix C4 solution before use.

1.1.1.10.1 Note: If more than 300 μl μL supernatant

was transferred at step 8, add 1.4 volumes of
Solution C4 to the supernatant and mix well by
pipetting.
1.1.1.11 Transfer the 700 μl mix to an AllPrep DNA
MinElute Spin Column placed in a 2 ml Collection
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Tube. Centrifuge at 8000 x g for 30 s at room

temperature.
1.1.1.12 After centrifugation, place the AllPrep DNA
MinElute Spin Column in a new 2 ml Collection Tube
and store it in a refrigerator at 2–8°C for DNA
purification.
Note:NOTE: Verify that no liquid remains on
top of the membrane. If the mixture has not
completely passed through the AllPrep
DNA MinElute Spin Column membrane,
centrifuge the spin column again at 13,000 x g
for 1 min.
1.1.1.12.1.1 Optional: If more than 300 μl μL
supernatant was transferred at step 8, the
AllPrep DNA MinElute Spin Column must be
loaded twice. First, transfer 700 μl of the
mix to the AllPrep DNA MinElute Spin
Column and centrifuge at 8000 x g for 30 s
at room temperature. After centrifugation,
place the DNA AllPrep Spin Column in a
clean 2 ml Collection Tube. Transfer the
remaining mix to the AllPrep DNA MinElute
Spin Column and centrifuge at 8000 x g for
30 s at room temperature. Place the AllPrep
DNA MinElute Spin Column in a clean 2 ml
Collection Tube and store in a refrigerator at
2–8°C for DNA purification.
1.1.2 Total RNA Purification
1.1.2.1 Add 700 μl 80% ethanol to the flow-through
from step 10, and mix well by pipetting. Do not
centrifuge.
1.1.2.1.1Note: Precipitates may be visible after addition
of ethanol. This does not affect the procedure.
1.1.2.1.1.1 Optional: If more than 300 μl supernatant
was transferred at step 8, add 1 volume of
80% ethanol to each of the two flow-
throughs. Mix well by pipetting.
1.1.2.2 Transfer up to 700 μl of the mix to an RNeasy
Mini Spin Column placed in a 2 ml Collection Tube.
Close the lid gently, and centrifuge at 8000 x g for
30 s. Discard the flow-through. Centrifuge
successive aliquots in the same RNeasy Mini Spin
Column. Discard flow-through after each
centrifugation. Reuse the 2 ml Collection Tube in
next step.
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1.1.2.3 Add 350 μl Buffer RW1 to the RNeasy Mini Spin

Column. Close the lid gently, and centrifuge at
>8000 x g for 30 s. Discard the flow-through. Reuse
the 2 ml Collection Tube in next step.
1.1.2.4 Add 500 μl Buffer RPE to the RNeasy Mini Spin
Column. Close the lid gently, and
centrifuge at 8000 x g for 30 s. Discard the flow-
through. Reuse the 2 ml Collection Tube in next
step.
1.1.2.5 Add 500 μl Buffer RPE to the RNeasy Mini Spin
Column. Close the lid gently and centrifuge at
>18,000 x g for 2 min. Discard the Collection Tube
and the flow-through.
1.1.2.6 Place the RNeasy Mini Spin Column in a clean 2
ml Collection Tube. Close the lid gently and
centrifuge at N18,000 x g for 1 min.
1.1.2.6.1Note: Perform this step to eliminate any
possible carryover of Buffer RPE or if residual
flow-through remains on the outside of the
RNeasy Spin Column.
1.1.2.7 Place the RNeasy Mini Spin Column in a clean
1.5 ml Collection Tube. Add 30 μl
RNase-Free Water directly to the Spin Column
membrane. Close the lid gently, and centrifuge at
8000 x g for 1 min.
1.1.2.8 Repeat last step for increased RNA yield.
1.1.3 Genomic DNA Purification
1.1.3.1 Add 500 μl Buffer AW1 to the AllPrep DNA
MinElute Spin Column from step 1.1.1.11. Close the
lid gently, and centrifuge at 8000 x g for 1 min.
Discard the flow-through. Reuse the 2 ml Collection
Tube in next step.
1.1.3.1.1Note: Verify that no liquid remains on top of
the membrane. If the mixture has not
completely passed through the AllPrep DNA
MinElute Spin Column membrane, centrifuge
again at 13,000 x g for 1 min.
1.1.3.1.2Optional: If RNA-free genomic DNA is required,
add 500 μl Buffer AW1 and 4 μl RNase A (25
mg/ml) to the AllPrep DNA MinElute Spin
Column. Incubate for 2 min at room
temperature before continuing with next step.
1.1.3.2 Add 500 μl Buffer AW2 to the AllPrep DNA
MinElute Spin Column. Close the lid gently and
centrifuge at 18,000 x g for 2 min.
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1.1.3.3 Place the AllPrep DNA MinElute Spin Column in

a clean 2 ml Collection Tube and discard the old
Collection Tube with the flow-through. Close the lid
gently, and centrifuge at 18,000 x g for 1 min.
1.1.3.3.1Note: Perform this step to eliminate any
possible carryover of Buffer AW2 or if residual
flow-through remains on the outside of the
AllPrep DNA MinElute Spin Column after last
step.
1.1.3.4 Place the AllPrep DNA MinElute Spin Column in
a clean 1.5 ml Collection Tube. Add 30 μl Buffer EB
directly to the spin column membrane, and close the
lid gently. Incubate at room temperature for 1 min,
then centrifuge at 8000 x g for 1 min.
1.1.3.5 Repeat last step for increased DNA yield.
1.2 Post-purification
1.2.1 Prepare Samples for storage
1.2.1.1 Centrifuge sample for 1 minute at 2000xg to
consolidate solution
1.2.1.2 Acquire two 1mL Cryovials for each sample
1.2.1.3 Pipet ~200uL sample into each of the two 1mL
cryovials.
NOTE: Pellet size will affect how much sample is
present, try for equal volume in both storage tubes
1.2.1.4 Short hand label tube tops with “Tough Spots”
Micro tube stickers and sample ID
ex. WG001 = 1
1.2.2 NanoDrop samples and record concentration, purity
1.2.2.1 Reference SOP #004 for NanoDrop procedure
1.2.3 Label each micro centrifuge tube
1.2.3.1 Open DYMO Label v.8 app on computer
KARNESLAB1
1.2.3.2 Select “Saved Labels” and open recent WARF
label
1.2.3.3 Update with sample ID, concentration, purity,
date, and initials of person who isolated DNA/RNA
1.2.3.4 Print first label
1.2.3.5 Add -1 to end of Sample ID to denote duplicate
1.2.3.6 Print second label
1.2.4 Place in Warfarin Gut Boxes in -80 C freezer

2. Quality Control

3. References
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1. AllPrep PowerFecal DNA/RNA Kit Handbook. Third Edition,

January 2018.
https://www.qiagen.com/us/resources/resourcedetail?
id=ed925da4-a8c4-49c9-adfd-c6004ae0bc2c&lang=en