Fecal DNA Extraction Kit

ISOFECAL
Manual (First edition)
Code No. 318-06271

NIPPON GENE CO., LTD.

ISOFECAL Manual (ver.1)
20120106KH

Table of contents

IProduct description

1

IIContents of kit

1

IIIStorage

2

IVPrecautions

2

VProtocol

2

VI< Reagents, instruments, etc., required in addition to this product >2< Standard protocol >3

Data collection

5

1. DNA extraction from fecal samples

5

2. Absorption spectra of fecal DNA

5

3. PCR of fecal DNA

6

VII Troubleshooting

6

ISOFECAL Manual (ver.1)
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Product description

ISOFECAL is a kit for extracting DNA from the fecal samples.
Using this kit, DNA is extracted by the heat extraction method in the presence of asurface-active agent. Thus, DNA extraction from microorganisms having strong cell walls
may be difficult. Use this kit for detecting Gram-negative bacteria such as Escherichia coli.
If fecal DNA reflecting more actual fecal microbiota needs to be extracted, use ISOFECAL forBeads Beating, which provides chemical lysis by a surface-active agent and physicaldisruption of cells by beads beating.

IIContents of kit

Lysis Solution F＊

50 ml × 1

Purification Solution＊

20 ml × 1

Precipitation Solution

40 ml × 1

Wash Solution

50 ml × 1

TE (pH8.0）

5 ml × 1

Ethachinmate

100 μl × 1

Manual×1*: White crystal deposition may take place in the Lysis Solution F and the Purification
Solution, but this will not affect quality or performance. In such cases, use after completely
dissolving the crystals by incubating the whole container at about 37-65°C (mixoccasionally).

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IIIStorage

All the reagents included in ISOFECAL can be stored at room temperature.
However, for the Precipitation Solution, the Wash Solution and the Ethachinmate, werecommend that care be taken to prevent contamination at the time of use (contamination byfungi and bacteria), with storage at a low temperature (2-10°C) after opening.

IVPrecautions• This product is a reagent for research and cannot be used for medical or other objectives.
• This product should be handled only by persons having basic knowledge of reagents.
• Handle this product in accordance with the descriptions in the manual.
• We are not responsible for problems caused if this product is not handled in accordancewith the manual.
• A patent has been filed for fecal DNA extraction with ISOFECAL by the University of TokyoTLO. Nippon Gene has been licensed to practice the fecal DNA extraction method by theUniversity of Tokyo TLO.

VProtocol< Reagents, instruments, etc., required in addition to this product >• 70% ethanol• Chloroform• Micropipette• Pipette tip• 2 ml microtube• Incubator• Microcentrifuge• Vortex mixer-2-ISOFECAL Manual (ver.1)
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< Standard protocol >(1) Put 0.2 g of fecal sample in a 2 ml Tube.
(2) Add 1 ml of Lysis Solution F.
(3) Suspend feces using a toothpick or the like, vortex for 1 min and then incubate at 65°C
for 1 hr. (Note 1)(4) Centrifuge (12,000 x g, 5 min, room temperature).
(5) Transfer 600 μl of the supernatant to a new tube, add 400 μl of Purification Solution, andmix well. (Note 2)(6) Add 600 μl of chloroform, vortex for 15 sec, and then centrifuge (12,000 x g, 15 min,room temperature).
(7) Transfer 800 μl of the aqueous layer to a new tube while taking care not to transfer theintermediate layer, add 800 μl of Precipitation Solution, mix well, and then centrifuge(20,000 x g, 15 min, 4°C). (Note 3)(8) Discard the supernatant, add 1 ml of Wash Solution, mix by inverting a few times, andthen centrifuge (20,000 x g, 10 min, 4°C). (Note 3), (Note 4)(9) Discard the supernatant, add 1 ml of 70% ethanol and 2 μl of Ethachinmate, vortex, andthen centrifuge (20,000 x g, 5 min, 4°C). (Note 3), (Note 5)(10) Discard the supernatant, air dry the precipitates and then dissolve the precipitates in 100
μl of TE (pH 8.0).

(Note 1) Confirm that the feces is completely suspended. Also, we recommend mixing
by vortexing or inverting every 10-20 min during the incubation.
(Note 2) Since feces absorbs the Lysis Solution F, 600 μl of the centrifuge supernatant
may not be recovered.

In such cases, scale down the steps (5)-(7) while

keeping the ratio of the solutions as it is.
Step (5), (6)Centrifuge supernatant : Purification Solution : Chloroform = 6 : 4 : 6
Step (7)Aqueous layer : Precipitation Solution = 1 : 1
No change after Step (8).
(Note 3) If the maximum centrifugal force of the available centrifuge is not more than
20,000 x g, then spin at the maximum centrifugal force (but not less than 12,000x g).

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(Note 4) Remove as much of the supernatant as possible.
It is possible that the substances included in the supernatant may inhibit PCR.
Also, when the yield of DNA is low, the precipitates are not visible in this step.
(Note 5) Fecal DNA can be recovered in a stable manner by adding Ethachinmate to 70%
ethanol. However, if Ethachinmate is not added, avoid vortexing and gently washthe precipitates, mixing by inversion.

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VI

Data collection

1. DNA extraction from fecal samples
1/200 amount of DNA extracted from 0.2 g of human fecal using this kit was electrophoresed
in 1% Agarose S.
123
Lane 1. OneSTEP Marker 1 (λ/ Hind III digest）
Lane 2. Fecal sample No.1
Lane 3. Fecal sample No.2

2. Absorption spectra of fecal DNA
Absorption spectra of DNA extracted from the human fecal using this kit was compared withthat of highly purified Lambda DNA (Code No.318-00414).

The fecal DNA extracted

0.4

with this kit was determined

λDNA0.3

to be high purity.

ISOFECAL

Abs

0.2

0.1

0-0.1
220

240

260

280

300

320340

Wavelength in nm-5-ISOFECAL Manual (ver.1)
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3. PCR of fecal DNA
Bacterial 16S rRNA gene fragments are amplified using the fecal DNA extracted with this kit
as the template, and 1/5 of the amount of the amplified product is electrophoresed in 2%Agarose S.
1VII2345678

Lane 1, 8. OneSTEP Ladder 100
Lane 2.

No Template Control

Lane 3.

Fecal DNA 16 pg

Lane 4.

Fecal DNA 80 pg

Lane 5.

Fecal DNA 400 pg

Lane 6.

Fecal DNA 2,000 pg

Lane 7.

Fecal DNA 10,000 pg

Troubleshooting
ProblemPossible cause
Few fecal
microorganisms

Possible countermeasure
Use fresh fecal whenever possible.
In step (9) of the protocol, the DNA precipitates are

Low yield of fecal
DNADNA
precipitates
may be washedaway.

prone to be peeled off. Use attached Ethachinmatetogether with 70% ethanol.
Also, since the precipitates become visible byEthachinmate, carefully remove the supernatant
while visually making sure the precipitates are notwashed away.

White crystal
deposits appear in
the Lysis Solution F.

Reagents are

Completely dissolve crystals by incubating at

precipitated

37-65°C and then use. This will not affect quality or

due to lowperformance.

temperature.

White crystal

Reagents are

Completely dissolve crystals by incubating at

deposits appear inprecipitated

37-65°C and then use. This will not affect quality or

the Purificationdue to low

performance.

Solution.

temperature.

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ProblemPossible cause

Possible countermeasure
Purchase a new kit. Since the composition of

Floating objects in Contamination

this solution allows the growth of fungi and thePrecipitation by fungi and the like, take thorough precautions against
contamination. We recommend storage at a low
Solutionlikethetemperature (2-10°C) after opening the package.
Purchase a new kit. Since the composition of
Floating objects inWash Solution

Contamination
by fungi and thelike

this solution allows the growth of fungi and thelike, take thorough precautions against
contamination.
We recommend storage at a low temperature(2-10°C) after opening the package.
Purchase new Ethachinmate.

Floating objects inEthachinmate

Contamination

Also, when using, take thorough precautions

by fungi and theagainst contamination.

likeWe recommend storage at a low temperature(2-10°C) after opening the package.

Lysis Solution F
spilling out of tube

The void volumeof the fecal islarge.

Reduce the amount of fecal sample to less than
0.2 g.

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NIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA
930-0834 JAPANTel+81-76-451-6548
URL
www.nippongene.com

ISOFECAL Manual (ver.1)
20120106KH