Soil DNA Extraction Kit

ISOIL for Beads Beating
Manual (Second edition)
Code No. 319-06201

NIPPON GENE CO., LTD.

ISOIL for Beads Beating Manual （ver.2）
110830KH

Table of contents
I

Product description

1

IIContents of kit

1

IIIStorage

2

IVPrecautions

2

V

Protocol2

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

2<Standard protocol>

3<Protocol to obtain maximum yield>

4

Data collection

5

1. DNA extraction from various soil samples

5

2. Absorption spectra of soil DNA

5

3. Detection of phyletic group by PCR

6

4. Restriction enzyme digestion of soil DNA

7

5. Incubation time at 65°C after Beads Beating and yield of soil DNA

8

6. PCR-DGGE analysis of soil DNA

9VII Troubleshooting

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I

Product description

ISOIL for Beads Beating is a kit for extracting DNA from the soil samples.
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, something which has been
considered difficult to achieve up to now.
ISOIL for Beads Beating uses both chemical lysis by a surface-active agent and physicaldisruption of cells by beads beating as the DNA extraction method. As a result, DNA can beextracted from microorganisms having strong cell walls, and soil DNA reflecting actual soil
microorganism mass structure can be obtained. Thus, the extracted soil DNA is suitable formass structure analysis of soil microorganisms, e.g., using PCR-DGGE analysis, soil
diagnosis, and estimation of soil biomass by quantitative determination of soil DNA.
However, users should be aware that DNA extracted by ISOIL for Beads Beating may havebeen subjected to physical shearing from beads beating.
Use ISOIL when extraction of high molecular weight soil DNA is required, e.g., when using
the soil DNA as a gene resource.

IIContents of kit

Beads Tubes

50 Tubes

Lysis Solution BB
Lysis Solution 20S

50 ml × 1＊

1.25 ml × 2

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* : Crystal deposition may take place in the Lysis Solution 20S 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 65°C (mixoccasionally).

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IIIStorage

All the reagents included in ISOIL for Beads Beating 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 soil DNA extraction with ISOIL for Beads Beating by theUniversity of Tokyo TLO. Nippon Gene has been licensed to practice the soil 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-ISOIL for Beads Beating Manual （ver.2）
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<Standard protocol>(1) Put 0.5 g of soil sample in a Beads Tube.
(2) Add 950 μl of Lysis Solution BB and 50 μl of Lysis Solution 20S.(Note 1)(3) Perform beads beating (4-6 m/sec or 4,200-6,800 rpm for 30-45 sec).(Note 2)(4) Centrifuge (12,000 x g, 1 min, room temperature).
(5) Transfer 600 μl of the supernatant to a new tube, add 400 μl of Purification Solution, andmix well.
(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 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) When DNA is extracted from soil containing a large quantity of allophones,
perform extraction using Lysis Solution BB SP1 (sold separately: Code No.
313-06221) in place of Lysis Solution BB.

Lysis Solution BB SP1 is an

optional solution exclusively for ISOIL for Beads Beating for extracting DNA
from the soil containing a large quantity of allophone.
(Note 2) Make sure that the lid of the Beads Tube is tightly sealed. A loose lid may
cause liquid to leak during beads beating.
(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
12,000 x g).
(Note 4) 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 5) Soil DNA can be recovered in a stable manner by adding Ethachinmate to 70%
ethanol. However, if Ethachinmate is not added, avoid vortexing and gently
wash the precipitates, mixing by inversion.
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<Protocol to obtain maximum yield>
This is a protocol for obtaining the maximum yield with ISOIL for Beads Beating. Use thismethod when the extraction yield of soil DNA is important, e.g., when estimating biomass
through quantitative measurement of soil DNA.
(1) Put 0.5 g of soil sample in a Beads Tube.
(2) Add 950 μl of Lysis Solution BB and 50 μl of Lysis Solution 20S prewarmed at 65°C.
(3) After performing beads beating (4-6 m/sec or 4,200-6,800 rpm for 30-45 sec), incubate
at 65°C for 30 min to 1 hr.
(4) Centrifuge (12,000 x g. 1 min, room temperature).
(5) The remaining steps are the same as steps (5)-(10) of the standard protocol (5)-(10).

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VI Data collection
1. DNA extraction from various soil samples
DNA was extracted with this kit from three kinds of soils, and the results indicated that DNA
could be extracted from any of these soils.
1234Lane 1. OneSTEP Marker 1 (λ/Hind III digest)
Lane 2. Control soil from the Yayoi agricultural field,
the University of Tokyo(allophane andosol soil/volcanic ash soil)
Lane 3. Saitama Agricultural Testing Station, agricultural
field soil (gray lowland soil/non-volcanic ash soil)
Lane 4. Hyogo Agricultural Testing Station, forest soil
(brown forest soil/non-volcanic ash soil)

One-twentieth of the amount of DNA extracted from 0.5 g of soil was electrophoresed
in 1% Agarose S.
2. Absorption spectra of soil DNA
Absorption spectra of DNA extracted from the control soil from the Yayoi agricultural field, theUniversity of Tokyo, using this kit was compared with that of highly purified Lambda DNA(Code No.318-00414).
0.4
ISOILfor Beads
BeatingDNA
Soil DNA
extracted
withで抽出した土壌
this kit.
LambdaLambdaDNADNA0.3

Abs

0.2
0.1

The soil DNA extracted

0with this kit was determined
to be high purity.

-0.1
220

240

260

280

300

320340

Wavelength in nm-5-ISOIL for Beads Beating Manual （ver.2）
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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.
123456712345671234567

Control soil from the Yayoi

Saitama Agricultural Testing

Hyogo Agricultural

agricultural field,Station, agricultural field soil

Testing Station, forest soil

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

Lane 1. OneSTEP Ladder 100
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）
Lane 7. Plants（597 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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4. Restriction enzyme digestion of soil DNA
Soil DNA extracted with this kit was digested with restriction enzyme EcoR I.
1234567Lane 1. OneSTEP Marker 1 (λ/Hind III digest)
Lane 2. Intact DNA of the control soil from the Yayoi
agricultural field, the University of Tokyo
Lane 3. DNA/EcoR I of the control soil from the Yayoi
agricultural field, the University of Tokyo
Lane 4. Intact DNA of agricultural field soil from Saitama
Agricultural Testing Station
Lane 5. DNA/EcoR I of agricultural field soil fromSaitama Agricultural Testing Station
Lane 6. Intact DNA of forest soil from Hyogo Agricultural
Testing Station
Lane 7. DNA/EcoR I of forest soil from Hyogo
Agricultural Testing Station-7-ISOIL for Beads Beating Manual （ver.2）
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5. Incubation time at 65°C after Beads Beating and yield of soil DNA
The relationship between the incubation time at 65°C after Beads Beating and the yield of soil
DNA was investigated, and the results indicated that in some cases the DNA yield wasincreased by incubating at 65°C.

12Allophane andosol soil
千葉農試森林土壌（アロフェン質黒ボク土壌）

Yield (μg-DNA/g-soil)
収量（ μg-DNA/g-soil ）10Gray lowland soil
埼玉農試畑土壌（灰色低地土）
兵庫農試森林土壌（褐色森林土）
Brown forest soil

86420015304560

インキュベート時間（分）Incubationtime (min)-8-ISOIL for Beads Beating Manual （ver.2）
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6. PCR-DGGE analysis of soil DNA
PCR-DGGE analyses were performed using the soil DNAs extracted with this kit.
123456Lane 1. Control soil from the Yayoi agricultural field, the University of Tokyo
Lane 2. Pasture land soil from Tanashi farm, the university of Tokyo
Lane 3. Forest soil from Chiba Agricultural Testing Station
Lane 4. Forest meadow soil from the Grass Field Testing Station
Lane 5. Agricultural field soil from Saitama Agricultural Testing Station
Lane 6. Forest soil from Hyogo Agricultural Testing Station
Data provided by: Hiroki Rai, Ph. D. Department of Applied Biological Chemistry,Graduate School of Agricultural and Life Sciences, The University of Tokyo-9-ISOIL for Beads Beating Manual （ver.2）
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VII Troubleshooting
ProblemPossible cause

Possible countermeasure
Use fresh soil whenever possible.

Few soilAlso, in somecases, DNA yield may increase by incubating at 65°C

microorganisms for 30 min to 1 hr after beads beating (see Datacollection 5)
In step (9) of the protocol, the DNA precipitates areLow yield of soil DNADNA

prone to be peeled off.

Use attached Ethachinmateprecipitates

together with 70% ethanol.

may be washedprecipitates become visible by Ethachinmate,away

carefully remove the supernatant while visuallyAlso, since themaking sure the precipitates are not washed away.
Extract using Lysis Solution BB SP1 (sold separately:
Too muchallophane in thesoil

Code No. 313-06221) in place of Lysis Solution BB.
Lysis Solution BB SP1 is an optional solution
exclusive for ISOIL for Beads Beating for extracting
DNA from soil containing a large quantity ofallophane.

DNA is sheared

When high molecular weight soil DNA is desired to be

Molecular weight ofby physicalextracted, use ISOIL, which employs the heatsoil DNA is low.

impact ofextraction method in the presence of a surface active

beads beating.

agent.

White crystal

Reagents are

Completely dissolve crystals by incubating at

deposits appear inprecipitated

37-65°C and then use.

the Lysis Solution

due to low

This will not affect quality or performance.

20S.

temperature.

White crystal

Reagents are

Completely dissolve crystals by incubating at

deposits appear inprecipitated

37-65°C and then use.

the Purification

due to low

This will not affect quality or performance.

Solution.

temperature.
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 a low temperature (2-10°C)
after opening the package.

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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 thelike

be 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 a low temperature(2-10°C) after opening the package.
Purchase 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 a low temperature(2-10°C) after opening the package.

Lysis Solution

The void volume

spilling out ofof the soil isBeads Tubelarge.

Reduce the amount of soil sample to less than
0.5 g.

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.

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NIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA 930-0834
JAPANTel076-451-6548
URL
https://www.nippongene.com/

ISOIL for Beads Beating Manual （ver.2）
110830KH