Fecal DNA Extraction Kit

ISOFECAL for Beads Beating
Manual (First edition)
Code No. 315-06281

NIPPON GENE CO., LTD.

ISOFECAL for Beads Beating Manual
20110830KH

Table of contents

I

Product 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

6VII Troubleshooting

7

ISOFECAL for Beads Beating Manual
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I

Product description

ISOFECAL for Beads Beating is a kit for extracting DNA from the fecal samples.
ISOFECAL for Beads Beating uses both chemical lysis by a surface-active agent andphysical disruption of cells by beads beating as the DNA extraction method. As a result,
DNA can be extracted from microorganisms having strong cell walls, and fecal DNA reflecting
actual fecal microflora can be obtained.
However, users should be aware that DNA extracted by ISOFECAL for Beads Beating may
have been subjected to physical shearing from beads beating.

IIContents of kitBeads Tubes
Lysis Solution F50 Tubes＊

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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Storage

All the reagents included in ISOFECAL for Beads Beating can be stored at room temperature.
However, for the Precipitation Solution, the Wash Solution and the Ethachinmate, we
recommend 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 for Beads Beating by theUniversity of Tokyo TLO. Nippon Gene has been licensed to practice the fecal DNA
extraction method by the University of Tokyo TLO.

VProtocol< Reagents, instruments, etc., required in addition to this product >• Beads Beating disruption apparatus (for 2 ml tube)• 70% ethanol• Chloroform• Micropipette• Pipette tip• 2 ml microtube• Incubator• Microcentrifuge• Vortex mixer-2-ISOFECAL for Beads Beating Manual
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<Standard protocol>(1) Put 0.2 g of fecal sample in a Beads Tube.
(2) Add 1 ml of Lysis Solution F.
(3) Perform beads beating (4-6 m/sec or 4,200-6,800 rpm for 30-45 sec).(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) Make sure that the lid of the Beads Tube is tightly sealed. A loose lid may cause
liquid to leak during beads beating.
(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

0.40.3

λDNAwith this kit was determined

ISOFECAL for Beads Beating

to be high purity.

0.2
Abs
0.1

0-0.1
220240260

280300320340

Wavelength in nm-5-ISOFECAL for Beads Beating Manual
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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.

12345678
Lane 1, 8. OneSTEP Ladder 100-6-

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

ISOFECAL for Beads Beating Manual
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VII

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 Ethachinmate
together with 70% ethanol.
Also, since the precipitates become visible byEthachinmate, carefully remove the supernatant
while visually making sure the precipitates are notwashed away.
When high molecular weight fecal DNA is desired

DNA is sheared
Molecular weight ofby physicalfecal DNA is low.

impact of beads
beating.

to be extracted, use ISOFECAL, which employs
the heat extraction method in the presence of asurface active agent.
However, please note that microorganisms having
strong cell walls may not be destroyed using
ISOFECAL.

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

the Purification

due to low

or performance.

Solution.

temperature.
Purchase a new kit. Since the composition of thisFloating objects inContamination

solution allows the growth of fungi and the like,the Precipitation

by fungi and the take thorough precautions against contamination.

Solutionlike

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

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ISOFECAL for Beads Beating Manual
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ProblemPossible cause

Possible countermeasure
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 againstcontamination.
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

The void volume

spilling out ofof the fecal isBeads Tubelarge.

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
https://www.nippongene.com/

ISOFECAL for Beads Beating Manual
20110830KH