Soil DNA Extraction Kit

ISOIL
Manual (Second edition)
Code No. 316-06211

NIPPON GENE CO., LTD.

ISOIL Manual（ver.2）
110830KH

Table of contents
I

Product description

1

II

Contents of kit

1

IIIStorage

2

IVPrecautions

2

VProtocol

2

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

2<Standard protocol>

3<Scale-up>

4

Data collection

5

1. DNA extraction from various soil samples

5

2. Size of soil DNA

5

3. Detection of phyletic group by PCR

6

4. Restriction enzyme digestion of soil DNA

7

5. Stirring during incubation at 65°C and yield of soil DNA

86. PCR-DGGE analysis of soil DNA

8VII Troubleshooting

9ISOIL Manual（ver.2）
110830KHIProduct description

ISOIL is a kit for extracting DNA from 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.
With ISOIL, DNA is isolated with a heat extraction method in the presence of asurface-active agent. Since soil DNA is extracted without applying physical force, highmolecular weight DNA can be obtained.

Therefore, soil DNA extracted with ISOIL is

suitable for applications involving gene resources, e.g., the construction of a metagenomiclibrary.
However, please note that microorganisms having strong cell walls may not be destroyed.
Please use ISOIL for Beads Beating, which uses disruption by beads beating to perform
group structure analysis of soil microorganisms through PCR-DGGE analysis andestimation of soil biomass by quantitative determination of soil DNA.

IIContents of kit

Lysis Solution HE

50 ml × 1

Lysis Solution 20S＊

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).

-1-ISOIL Manual（ver.2）
110830KH

IIIStorage

All the reagents included in ISOIL 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.

IV•

Precautions

This product is a reagent for research and cannot be used for medical or otherobjectives.

•This product should be handled only by persons having a 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 by the University of Tokyo TLO.
Nippon Gene has been licensed to practice the soil 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-ISOIL Manual（ver.2）
110830KH

< Standard protocol >(1) Put 0.5 g of soil sample in a 2 ml tube.
(2) Add 950 μl of Lysis Solution HE and 50 μl of Lysis Solution 20S, and, after mixing wellby inversion, incubate at 65°C for 1 h.(Note 1)(3) Centrifuge (12,000 x g, 1 min, room temperature).
(4) Transfer 600 μl of the supernatant to a new tube, add 400 μl of Purification Solution,and mix well.
(5) Add 600 μl of chloroform, vortex for 15 sec, and then centrifuge (12,000 x g, 15 min,room temperature).
(6) 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 2)(7) 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 2),(Note 3)(8) 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 2),(Note 4)(9) Discard the supernatant, air dry the precipitates and then dissolve the precipitates in
100 μl of TE (pH 8.0).

(Note 1) The yield of soil DNA is increased by mixing by inversion in the middle of theprocess (see the data collection 5). If a rotator or the like is available, werecommend incubating while gently rotating.
(Note 2) 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).
(Note 3) 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 (7) can be skipped.
(Note 4) Soil DNA can be recovered in a stable manner by adding Ethachinmate to 70%
ethanol.

However, if for some reason Ethachinmate is not added, avoid

vortexing and gently wash the precipitates, mixing by inversion.

-3-ISOIL Manual（ver.2）
110830KH

< Scale-up >Scale-up of soil DNA extraction with ISOIL is easy. We recommend scale-up extraction
when a large amount of high molecular weight soil DNA is required for cloning, constructionof a library, or the like. The following points should be noted for scale-up.
1. The ratio of Lysis Solution HE and Lysis Solution 20S must be 95 : 5.
2. The ratio of the supernatant of the centrifugation at step (4) of the standard protocol
and the Purification Solution must be 6 : 4.
3. The chloroform to be added must be equal to the amount to the supernatant fromthe centrifugation at step (4) of the standard protocol.
4. The Precipitation Solution to be added must be equal to the amount of the aqueouslayer recovered after the chloroform treatment.

Example of scale-up: DNA extraction from 5 g of soil (ten-fold scale-up)(1) Put 5 g of soil sample in a 50 ml centrifuge tube.
(2) Add 9.5 ml of Lysis Solution HE and 0.5 ml of Lysis Solution 20S, mix well by inversion
and then incubate at 65°C for 1 hr while gently shaking.
(3) Centrifuge (5,000 x g, 10 min, 25°C).
(4) Transfer 6 ml of the supernatant to a new tube, add 4 ml of Purification Solution, andmix well.
(5) Add 6 ml of chloroform, mix well by inversion until homogeneous, and then centrifuge(5000 x g, 15 min, 4°C).
(6) Carefully transfer 8 ml of the aqueous layer to a new tube so that no intermediate layer
contaminates the aqueous layer, mix well with 8 ml of Precipitation Solution, andcentrifuge (9,000 x g, 30 min, 4°C).
(7) Discard the supernatant, add 10 ml of Wash Solution, mix by inverting a few times, andcentrifuge (9,000 x g, 10 min, 4°C).
(8) Discard the supernatant, add 10 ml of 70% ethanol and 10 μl of Ethachinmate, mix byinversion, and then centrifuge (9,000 x g, 10 min, 4°C).(Note 1)(9) Discard the supernatant, and, after air-drying, dissolve the precipitates in 500 μl of TE(pH8.0).
(Note 1) 1-2 μl of Ethachinmate is sufficient for 100 μl of the final solution. Adding too
much Ethachinmate increases the viscosity and may cause problems in
subsequent operations.

-4-ISOIL Manual（ver.2）
110830KH

IV Data collection
1. DNA extraction from various soil samples
DNA was extracted by using this kit from three kinds of soils, and the results indicated thatDNA could be extracted from any of these soils.
1234
Lane 1. OneSTEP Marker 1 (λ/Hind III digest)
Lane 2. Control soil from the Yayoi agricultural field, theUniversity of Tokyo (allophone andosol soil/volcanicash soil)
Lane 3. Saitama Agricultural Testing Station, agricultural fieldsoil (gray lowland soil/non-volcanic ash soil)
Lane 4. Hyogo Agricultural Testing Station, forest soil (brownforest soil/non-volcanic ash soil)

1/10 amount of DNA extracted from 0.5 g of soil was electrophoresed in 1% Agarose S.

2. Size of soil DNA
Soil DNA extracted using this kit was electrophoresed in 0.5% Agarose H. The results
indicated that the extracted soil DNA was not less than 50 kb.
12Lane 1. Marker 7GT (166, 50, 42, 23, 21, 18, 10, 1.3 kb)
Lane 2. DNA extracted from the control soil from the Yayoi agricultural
field, the University of Tokyo-5-

ISOIL Manual（ver.2）
110830KH

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.
12345671234567123456Soil from the Yayoi agricultural field,

Saitama Agricultural

Hyogo Agricultural Testing

the University of Tokyo

Testing Station, agricultural

Station, forest soil

(allophone andosol soil)

field soil (gray lowland soil)(brown forest soil)

7

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
and Spores in Soil.
Kuske CR, Banton KL, Adorada DL, Stark PC, Hill KK, Jackson PJ.
Appl Environ Microbiol. 1998 Jul 1;64(7):2463-72.

-6-ISOIL Manual（ver.2）
110830KH

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 theYayoi
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 from
Saitama 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 Manual（ver.2）
110830KH

5. Stirring during 65°C incubation and yield of soil DNA
The relationship between stirring during incubation at 65°C and the yield of soil DNA wasinvestigated, and the results indicated that the DNA yield was increased by performing
mixing by inversion during incubation.
12345
Lane 1. OneSTEP Marker 1 (λ/Hind III digest）
Lane 2. Letting stand for 60 min
Lane 3. Mixing by inversion at 30 min intervals
Lane 4. Mixing by inversion at 20 min intervals
Lane 5. Mixing by inversion at 10 min intervals

6. PCR-DGGE analysis of soil DNA
PCR-DGGE analyses were performed using the soil DNAs extracted with this kit.
123456
Lane 1. Control soil from the Yayoi agricultural field, theUniversity of Tokyo
Lane 2. Pasture land soil from Tanashi farm, the University ofTokyo
Lane 3. Forest soil from Chiba Agricultural Testing Station
Lane 4. Forest meadow soil from the Grass Field TestingStationLane 5. Agricultural field soil from Saitama Agricultural TestingStationLane 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-8-ISOIL Manual（ver.2）
110830KH

VII Troubleshooting

ProblemPossible cause

Possible countermeasure
Use fresh soil whenever possible.

Few soilAlso, DNA yield may increase by mixing themicroorganisms

solution by inversion at 10 min intervals while
incubating at 65°C (see data collection 5).
In the step (8) of the protocol, the DNA
precipitates are prone to be peeled off.

UseDNA precipitates

Ethachinmate together with 70% ethanol. Also,

Low yield of soil

may be washedsinceDNAaway.

Ethachinmate, carefully remove the supernatanttheprecipitatesbecomevisibleby

while visually making sure the precipitates are
not washed away.
Use ISOIL for Beads Beating which employs
Microorganisms

physical disruption of microorganisms body by

are not wellbeads beating.

disrupted.

shearing force is applied, the extracted soil DNA

However, since the physicalis about 23 kb.
PhysicalShearing of DNA

shearing occurs
during pipetting
and stirring.

Cut off the ends of pipette tips.

Also, whenstirring the solution, avoid using a vortex and mix
well by inversion.

White crystal

Reagents are

Completely dissolve crystals by incubating at

deposits appear inprecipitated due

37-65°C and then use.

the Lysis Solution

to a low

quality or performance.

20S.

temperature.

White crystal

Reagents are

Completely dissolve crystals by incubating at

deposits appear inprecipitated due

37-65°C and then use.

the Purification

to a low

quality or performance.

Solution.

temperature.

-9-This will not affectThis will not affect

ISOIL Manual（ver.2）
110830KH

ProblemPossible cause

Possible countermeasure
Purchase a new kit.
Since the composition of this solution allows theFloating objects in
the Precipitation
Solutiongrowth of fungi and the like, take thoroughContamination byprecautions against contamination.

fungi and the like

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

Purchase a new kit.
However,ifthecontamination

of

humic

substance is minor, step (8) of the protocol canbe skipped. In such cases, the Wash Solution is
Floating objects in Contamination bynot required.

Wash Solution

Since the composition of this solution allows thefungi and the likegrowth 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.
Floating objects in Contamination byEthachinmate

fungi and the likeAlso, when using, take thorough precautions
against contamination.
We recommend storage at a 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 with this kit DNA can be extracted fromdozens of various Japanese soil types. However, since soil can vary greatly, the yield may
not be high 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 particularsoil sample has unique characteristics which other samples do not have rather than aproblem with the reagents and procedures.

- 10 -ISOIL Manual（ver.2）
110830KH

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

ISOIL Manual（ver.2）
110830KH