Soil DNA Extraction Kit

ISOIL Large for Beads ver.2
Manual (First edition)

Code No. 312-06791

NIPPON GENE CO., LTD.

ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

Table of contents
I

Product description

1

II

Contents of kit

1

III

Storage

2

IV

Precautions

2

V

Protocol2

VI<Reagents, instruments, etc., required in addition to this product>

2<Standard protocol>

3<Optional protocol① Repurification of soil DNA>

5

<Optional protocol② Scaling up to recover a minute amount of DNA>

6

Data collection

8

1. DNA extraction from various soil samples

8

2. Absorption spectra of soil DNA

8

3. Detection of phyletic group by PCR

9

4. Repurification of soil DNA

10

VII Troubleshooting

11

ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

I

Product description

ISOIL Large for Beads ver.2 is a kit for extracting DNA from soil sample of 5 g.
By using an extracting solution with a special composition, DNA can be extracted not only
from non-volcanic ash soil but also from volcanic ash soil, which has been considered difficult
to achieve up to now.
In the protocol of ISOIL Large for Beads ver.2, DNA is extracted by thermal extraction method
under the presence of surface-active agent and physical disruption of cells by beads beating.
Also, by precipitating DNA under special conditions, highly purified soil DNA can be extracted
at a higher concentration compared to the conventional method.

II

Contents of kit(For 8 reactions)
Beads Tubes

8 tubes

Lysis Solution BB
Lysis Solution 20S

80 ml × 1＊

4 ml × 1

Purification Solution＊

45 ml × 1

Precipitation Solution

90 ml × 1

Wash Solution

45 ml × 1

TE (pH 8.0）

10 ml × 1

Ethachinmate

100 μl × 1

Manual×1* : Crystal may form in the Lysis Solution 20S and in the Purification Solution, but this will notaffect quality or performance. In such cases, use after completely dissolving the crystals
by incubating the whole container at about 37-65°C (mix occasionally).

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ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

III

Storage

All the reagents included in ISOIL Large for Beads ver.2 can be stored at room temperature.
Care must be taken to protect the Precipitation Solution, the Wash Solution, and theEthachinmate from contamination at time of use, and it is recommended to store at low
temperature (2-10°C) after opening.

IV

Precautions• 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 soil DNA extraction with ISOIL Large for Beads ver.2 by the
University of Tokyo TLO. Nippon Gene has been licensed to practice the soil DNA
extraction method by the University of Tokyo TLO.

V

Protocol< Reagents, instruments, etc., required in addition to this product >• 70% ethanol• Chloroform• Messpipette• Micropipette• Pipette tip• Centrifuge tube
• Vortex mixer• Incubator• Centrifuge-2-

ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

<Standard protocol>(1) Put 5 g of soil sample in a Beads Tube.
(2) Add 9.5 ml of Lysis Solution BB and 0.5 ml of Lysis Solution 20S.
(3) After vortexing at the highest speed for 10 min, incubate at 65°C for 60 min. (Note 1) (Note 2)(Note 3)(4) Centrifuge (4,000 x g, 5 min, room temperature).
(5) Transfer 6 ml of the supernatant to a new centrifuge tube, add 4 ml of Purification
Solution, and mix well. (Note 4)(6) Add 6 ml of chloroform, vortex for 1 min, and then centrifuge (4,000 x g, 15 min, room
temperature). (Note 1)(7) Transfer the aqueous layer (upper layer) to a new centrifuge tube while taking care not totransfer the intermediate layer, add an equal volume of the Precipitation Solution, mix
well and centrifuge (8,000 x g, 60 min, 4°C). (Note 5) (Note 6)(8) Discard the supernatant, rinse the precipitates and the wall of the centrifuge tube byadding 5 ml of the Wash Solution, and then centrifuge (8,000 x g, 10 min, 4°C). (Note 6) (Note7) (Note 8)(9) Discard the supernatant, add 5 ml of 70% ethanol and 10 μl of Ethachinmate, vortex, andthen centrifuge (8,000 x g, 10 min, 4°C). (Note 6) (Note 8) (Note 9)(10) Discard the supernatant, air dry the precipitates and then dissolve the precipitates in an
appropriate amount of TE(pH 8.0) (about 0.3-1 ml). (Note 10)(Note 1)Make sure that the lid of the centrifuge tube is tightly closed so as not to cause
leak.

(Note 2)The yield of soil DNA is increased by mixing by inversion during the incubation.

(Note 3)

When the beads disruption apparatus for 50 ml centrifuge tubes is usedinstead of the vortex, the beating time may be shortened.

Optimize

conditions (speed, time).
(Note 4) When the water content of the soil sample is low (when the sample is dry), thevolume of the supernatant after the centrifugation (4) may be less than 6 ml.
In such cases, continue the experiment by adjusting the ratio among thevolume of the supernatant after the centrifugation, the volume of the
Purification Solution and the volume of chloroform to 3 : 2 : 3. This volume
ratio is very important.
(Note 5) When the supernatant after the centrifugation of (4) is 6 ml, the Purification
Solution is 4 ml and chloroform is 6 ml, the aqueous phase (upper layer)
after the centrifugation (6) will be a little more than 8 ml. Carefully collect 8 ml

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ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

so that the intermediate layer is not mixed, and proceed to the next step.
(Note 6)

At Nippon Gene, centrifugation is performed at 8,000 x g for 60 min using
conical disposable centrifuge tubes and a corresponding centrifuge rotor.
Make sure to set the centrifugal force to be not more than the allowablemaximum centrifugal force of the centrifuge tubes to be used. Also, adjust
the centrifugal force and time in accordance with the centrifuge tubes to be
used.

(Note 7)

Normally DNA is not visible at this step. Take care not to touch the wall of the
tube where DNA may be precipitated with a pipette or the like.

The

precipitates become visible at (9) after addition of Ethachinmate and vortexing.
(Note 8)Remove as much of the supernatant as possible. The colored substance(humic substance) in the supernatant is known to inhibit PCR.

Also, ifcontamination by humic substance is minimal (less coloring of the aqueous
layer after the chloroform treatment), step (8) can be skipped.
(Note 9)

Soil DNA can be recovered in a stable manner by adding Ethachinmate to
70% ethanol.

(Note 10) When the obtained soil DNA is colored or PCR inhibition is observed, performthe repurification of the soil DNA by the optional protocol① described below.

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ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

<Optional protocol① Repurification of soil DNA>
When the obtained soil DNA is colored or PCR inhibition is observed, perform therepurification of the soil DNA by the following protocol.
(1) Transfer 300 μl of the soil DNA obtained by the standard protocol and optional protocol②
to a new 1.5 ml tube, add 200 μl of Purification Solution, and mix well.
(2) Add 300 μl of chloroform, vortex for 15 sec, and then centrifuge (12,000 x g, 15 min, room
temperature).
(3) Transfer 400 μl of the aqueous layer (upper layer) to a new 1.5 ml tube while taking care
not to transfer the intermediate layer, add 400 μl of Precipitation Solution, mix well, andthen centrifuge (20,000 x g, 10 min, 4°C).
(4) Discard the supernatant, rinse the precipitates and the wall of the tube by adding 500 μl of
the Wash Solution, and then centrifuge (20,000 x g, 10 min, 4°C). (Note 1) (Note 2)(5) Discard the supernatant, add 500 μl of 70% ethanol and 1 μl of Ethachinmate, vortex, andthen centrifuge (20,000 x g, 5 min, 4°C). (Note 2) (Note 3)(6) Discard the supernatant, air dry the precipitates and then dissolve the precipitates in an
appropriate amount of TE(pH 8.0) (about 100-300 μl).

(Note 1)

Normally DNA is not visible at this step. Takecare not to touch the wall of the tube where DNA may be precipitated with a pipette or the
like. The precipitates become visible at (5) after addition of Ethachinmate
and vortexing.
(Note 2)

Remove as much of the supernatant as possible. The colored substance(humic substance) in the supernatant is known to inhibit PCR.

Also, ifcontamination by humic substance is minimal (less coloring of the aqueous
layer after the chloroform treatment), step (4) can be skipped.
(Note 3)

Soil DNA can be recovered in a stable manner by adding Ethachinmate to
70% ethanol.

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ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

<Optional protocol② Scaling up to recover a minute amount of DNA>
When the amount of microorganisms included in a soil sample is small and a sufficientamount of DNA for analysis is thought to be difficult to recover from 5 g of the soil sample,
extract DNA from 20 g of the sample by the following protocol.
(1) Put 5 g each of the soil samples in 4 Beads Tubes (20 g total).
(2) Add 9.5 ml of Lysis Solution BB and 0.5 ml of Lysis Solution 20S to each tube.
(3) After vortexing at the highest speed for 10 min, incubate at 65°C for 60 min. (Note 1) (Note 2)(Note 3)(4) Centrifuge (4,000 x g, 5 min, room temperature).
(5) Transfer 6 ml each of the supernatant to 4 new 50 ml tubes so that no precipitates are
mixed, and add 4 ml of the Purification Solution to each tube and mix well. (Note 4)(6) Add 6 ml of chloroform to each tube, vortex for 1 min, and then centrifuge (4,000 x g, 15min, room temperature). (Note 1)(7) Transfer the aqueous layer (upper layer) from 2 tubes to a new 50 ml tube while taking
care not to transfer the intermediate layer, add an equal volume of the Precipitation
Solution to each tube, mix well and centrifuge (8,000 x g, 60 min, 4°C). (Note 5) (Note 6)(Combine the sample from 4 tubes into 2 tubes).
(8) Discard the supernatant, rinse the precipitates and the wall of the centrifuge tube byadding 5 ml of the Wash Solution to each tube, and then centrifuge (8,000 x g, 10 min,4°C). (Note 6) (Note 7) (Note 8)(9) Discard the supernatant, add 5 ml of 70% ethanol and 10 μl of Ethachinmate to eachtube, vortex, and then centrifuge (8,000 x g, 10 min, 4°C). (Note 6) (Note 8) (Note 9)(10) Discard the supernatant, air dry the precipitates and then dissolve the precipitates in an
appropriate amount of TE(pH 8.0) (about 0.3-1 ml). (Note 10)(Note 1)Make sure that the lid of the centrifuge tube is tightly closed so as not to cause
leak.

(Note 2)The yield of soil DNA is increased by mixing by inverting during the incubation.

(Note 3)

When the beads disruption apparatus for 50 ml centrifuge tubes is usedinstead of the vortex, the beating time may be shortened.

Optimize

conditions (speed, time).
(Note 4) When the water content of the soil sample is low (when the sample is dry), thevolume of the supernatant after the centrifugation (4) may be less than 6 ml.
In such cases, continue the experiment by adjusting the ratio among thevolume of the supernatant after the centrifugation, the volume of the-6-

ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

Purification Solution and the volume of chloroform to 3 : 2 : 3. This volume
ratio is very important. If the supernatant of (4) after the centrifugation ismuch greater, about 7.5 ml may be recovered.
(Note 5) When the supernatant after the centrifugation of (4) is 6 ml, the Purification
Solution is 4 ml and chloroform is 6 ml, the aqueous phase (upper layer) after
the centrifugation (6) will be a little more than 8 ml. Carefully transfer 16 ml of
the aqueous phase (collected from 2 tubes) to a new 50 ml tube so that theintermediate layer is not mixed.
(Note 6)

At Nippon Gene, centrifugation is performed at 8,000 x g for 60 min using
conical disposable centrifuge tubes and a corresponding centrifuge rotor.
Make sure to set the centrifugal force to be not more than the allowablemaximum centrifugal force of the centrifuge tubes to be used. Also, adjust
the centrifugal force and time in accordance with the centrifuge tubes to be
used.

(Note 7)

Normally DNA is not visible at this step. Take care not to touch the wall of the
tube where DNA may be precipitated with a pipette or the like.

The

precipitates become visible at (9) after addition of Ethachinmate and vortexing.
(Note 8)Remove as much of the supernatant as possible. The colored substance(humic substance) in the supernatant is known to inhibit PCR.

Also, ifcontamination by humic substance is minimal (less coloring of the aqueous
layer after the chloroform treatment), step (8) can be skipped.
(Note 9)

Soil DNA can be recovered in a stable manner by adding Ethachinmate to
70% ethanol.

(Note 10) When the obtained soil DNA is colored or PCR inhibition is observed, performthe repurification of the soil DNA by the optional protocol① described above.

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ISOIL Large for Beads ver.2 Manual （ver.1）
130426KHVI Data collection
1. DNA extraction from various soil samples
DNA was extracted with this kit from two kinds of soils, and the results indicated that DNA
could be extracted from any of these soils.
123
Lane 1. OneSTEP Marker 1 (λ/Hind III digest)
Lane 2. Control soil from the Yayoi agricultural field,
the University of Tokyo(allophone andosol soil/volcanic ash soil)
Lane 3. Experimental field, rice paddy soil, the Akita
Prefectural University(gray lowland soil/non-volcanic ash soil)

1/200 of the amount of DNA extracted from 5 g of soil was electrophoresed in 1% Agarose S.
2. Absorption spectra of soil DNA
Absorption spectra of DNA extracted from the soil using this kit was compared with highly
purified Lambda DNA (Code No.318-00414).

0.4

Sample A:
高純度に精製された
LambdaDNA
Lambda DNA (Code No.
318-00414)
東京大学
Sample A弥生圃場 対照区 (黒ボク土)
Sample B
秋田県立大学実験圃場 水田 (灰色低地土)

0.3

Control soil from the Yayoi agricultural
field, the University of Tokyo(allophone andosol soil/volcanic ash soil)

0.1

Sample B:

Abs

0.2

Experimental field, rice paddy soil, the0

Akita Prefectural University(gray lowland soil/non-volcanic ash soil)

-0.1
220

240

260

280

300

320

340

Wavelength in nm-8-

ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH3. Detection of phyletic group by PCR
Soil DNA extracted with this kit was analyzed by PCR using primers for phyletic groupdetection, and the results indicated that DNA fragments derived from various phyletic groups
were detected.
1

2

3

4

5

6

1

2

3

4

5

6

Control soil from the Yayoi

Experimental field, rice paddy soil,

agricultural field,

the Akita Prefectural University

the University of Tokyo(gray lowland soil)(allophone andosol soil)

Lane 1. OneSTEP Ladder 100 (0.1-2 kb)
Lane 2. Bacteria（723 bp）
Lane 3. Bacillus species and relatives（600 bp）
Lane 4. High-G+C gram-positive bacteria（542 bp）
Lane 5. Streptomyces species and related taxa（1,243 bp）
Lane 6. Fungi, protists, and green algae（555 bp）
A portion of the PCR product was electrophoresed in 2% Agarose S.

Reference for primers:
Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells andSpores in Soil.
Kuske CR, Banton KL, Adorada DL, Stark PC, Hill KK, Jackson PJ.
Appl Environ Microbiol. 1998 Jul 1; 64(7): 2463-72.

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ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

4. Repurification of soil DNA
Soil DNA purified from andosol soil with high humic substance content by the “standard
protocol” is repurified by the “Optional protocol① Repurification of soil DNA” and theabsorption spectra were compared.

Controlsoil fromthe Yayoi agricultural
field,
東京大学
弥生圃場
対照区
the University of Tokyo

0.4

高純度に精製された
LambdaDNA
Lambda DNA (Code No.
318-00414)
Standard protocol <one purification>
標準プロトコル（精製1回）
Optional protocol① <two purifications>
オプションプロトコル①（精製2回）

0.3

0.2

The soil DNA purified by theStandard protocol was found tohave slight coloration, but colorlessAbs

clear solution was obtained byrepurifying by the Optional protocol

0.1

①.
0

-0.1
220

240

260

280

300

320

340

Wavelength in nm

Agricultural
field in the center crop field
東北農業研究センター内圃場
畑(National Agricultural Research Center for Tohoku Region)

0.4

高純度に精製された
LambdaDNA
Lambda DNA (Code No.
318-00414)
Standard protocol <one purification>
標準プロトコル（精製1回）
Optional protocol① <two purifications>
オプションプロトコル①（精製2回）

0.3

0.2

The soil DNA purified by theStandard protocol was found tohave pale brown coloring, but

Abscolorlessclear

solutionwasobtained by repurifying by the

0.1

Optional protocol①.
0

-0.1
220

240

260

280

300

320

340

Wavelength in nm

By performing repurification, the DNA was purified to a higher degree than any other soil DNA.
Also, the yield in this repurification was around 70%.

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130426KH

VII Troubleshooting
ProblemPossible cause
Few soil

Possible countermeasure
Use fresh soil whenever possible.

microorganisms
In step (9) of the protocol, the DNA precipitates are

Low yield of soil DNA

DNA

prone to be peeled off.

Use attached Ethachinmate

precipitates

together with 70% ethanol.

may be washedprecipitates become visible by Ethachinmate,away

carefully remove the supernatant while visually

Also, since themaking sure the precipitates are not washed away.
The

For some soil, which does not adsorb DNA such asconcentration

gray lowland soil and brown forest soil, it is knownof Lysisthat DNA yield can be increased by using the Lysis

Solution BB is

Solution BB diluted two fold with distilled water in

not optimal.

place of the Lysis Solution BB.

DNA is sheared

When high molecular weight soil DNA is desired to be

Molecular weight ofby physicalextracted, use ISOIL, which employs the thermal

soil DNA is low.

shock byextraction method in the presence of a surface active

beads crush.

agent.

White crystals

Completely dissolve crystals by incubating at

deposit appear in the

Reagents are

37-65°C and then use.

Lysis Solution BB,

precipitatedThis will not affect quality or performance.

Lysis Solution 20S

due to low

and Purification

temperature.

Solution.
Purchase a new kit.

Since the composition of thisFloating objects inContamination

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

by fungi and the thorough precautions against contamination. We

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

- 11 -ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH

ProblemPossible cause

Possible countermeasure
Purchase a new kit.
However, if the contamination of humic
substance is minor, step (8) of the protocol can

Floating objects in
Wash Solution

Contamination
by fungi and thelikebe skipped. In such cases, the Wash Solution is
not required.
Since the composition of this solution allows thegrowth of fungi and the like, take thoroughprecautions against contamination.
We recommend storage at low temperature(2-10°C) after opening the package.
Purchase a new Ethachinmate.
However, when DNA yield is not important,

Floating objects inEthachinmate

Contamination

Ethachinmate is not needed.

by fungi and the Also, when using, take thorough precautions
likeagainst contamination.
We recommend storage at low temperature(2-10°C) after opening the package.

Soil is composed of inorganic substances such as various minerals and clay, soil organic
substances including various plants, dead plants and humic substance, and various
microorganisms living therein. Therefore, it can be said that every soil is different.
Our test results until now have confirmed that DNA can be extracted with this kit from dozens
of various Japanese soil types. However, since soil can vary greatly, the yield may not behigh in some samples depending on the type and condition of the soil.
If the yield of soil DNA extracted with this kit is low, the problem may be that the particular soil
sample has unique characteristics which other samples do not have rather than a problem
with the reagents and procedures.

- 12 -ISOIL Large for Beads ver.2 Manual （ver.1）
130426KHNIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA 930-0834
JAPAN
Tel
+81-76-451-6548
URL
https://www.nippongene.com/

ISOIL Large for Beads ver.2 Manual （ver.1）
130426KH