DNA extraction method using sodium iodide without phenol and chloroform

DNA Extractor® Kit Series
The sodium iodide method is introduced in USP 40 - NF 35 2nd "NUCLEIC ACID BASED TECHNIQUES - APPROACHES FOR DETECTING TRACE NUCLEIC ACIDS(RESIDUAL DNA TESTING)".

A widely used method to extract DNA from tissues derived from living body is to precipitate and collect DNA using phenol
and chloroform. However, this method requires the use of phenol and chloroform, hazardous substances.
This DNA extraction method uses sodium iodide known as a chaotropic agent instead of these organic solvents.
Sodium iodide has protein denaturation action and solubilization action. Sodium iodide releases chaotropic ions (I-) into
the solution; the ions increase water solubility of hydrophobic molecules in the sample, and its action to weakenhydrophobic bond contributes particularly to denaturation and solubilization of membrane proteins such as nuclear membrane.
In addition, no solid phase extraction using silica carrier inhibits DNA loss by adsorption; moreover, the entire operation is
performed in a single tube, realizing a high yield rate.

Features□ Organic solvents such as hazardous phenol and chloroform are not required.
□ The entire process is performed in a single tube, causing no loss, contamination, or mechanical DNA
damage in operation.
□ No solid phase extraction using silica carrier causes no loss by adsorption.
□ Little oxidation damage to DNA and mechanical DNA damage□ A high yield rate is realized.
1□ The product series used in the accumulated total of 963 publications ＊＊ 1: Source: Google scholar

List of DNA Extractor® Kit series using the sodium iodide method02

SampleProduct Name

Features

Viral DNA in serum/biological products

DNA Extractor® Kit

Low-cost, extraction of trace amount of DNA

Whole blood (human, cow, horse),
cultured cells, tissues

DNA Extractor® WB Kit

Can extract high-molecular DNA

Genomic DNA in serum/plasma

DNA Extractor® SP Kit

Can remove blood-derived lipids

Oxidative stress marker 8-OHdG DNA

DNA Extractor® TIS Kit

Inhibits DNA oxidation in extraction process

Body hairs, nails, blood stains,
saliva stain (forensic samples)

DNA Extractor® FM Kit

DNA extraction from trace amount of samples

Extraction of residual DNA in biological products and viral
DNA in serum

DNA Extractor® Kit

Wako Cat. No.

295-50201

This kit is intended for use in extracting host cell-derived
residual DNA remaining in biological components or viral
DNA in serum using the Sodium Iodide method.
The extracted DNA can be quantified using qPCR or theThreshold Immunoassay System® provided by Molecular
Devices, LLC.
Using DNA Extractor® kit is recommended for testing andmanagement of the amount of DNA derived from host cells
such as CHO cells, E. coli and yeast.

Extraction from serum

Package Size

50 tests

Operation time: 1 to 1.5 hours
Sample amount: 100 μL/test

Features□ Infinitesimal amount of DNA (10 pg) can also be collected
at a high yield of about 90%.
□ Organic solvents such as phenol and chloroform are notrequired.
□ The entire operation is completed in a single tube.

□ The operation is completed in a short time (1 to 1.5
hours).

Sample(100μL)

Kit Contents [50 tests (sample 100 μL)]:

Sodium N-Lauroyl Sarcosinate Solution /Glycogen Solution / Sodium Iodide Solution 300μL

1. Sodium iodide solution..................................................... 26 mL × 1

60℃, 15 minutes

2. Sodium N-Lauroyl sarcosinate solution....................... 1.2 mL × 1

Isopropanol 400μL

3. Washing solution (A)......................................................... 42 mL × 1

Room temperature, 15 minutes

4. Washing solution (B)......................................................... 40 mL × 2

10,000 × g, room temperature, 15 minutes

5. Glycogen solution............................................................. 0.1 mL × 1

Precipitate Remove the supernatant
Washing Solution (B) / Glycogen Solution 1mL

Principle of DNA extraction

10,000 × g, room temperature, 5 minutei. Proteins	and lipids in samples are made soluble with sodium
iodide and surfactant.

Precipitate Remove the supernatant
Dry under reduced pressureii. 	Then, isopropanol is added to the samples to co-precipitate
DNA with glycogen.

DNAiii.	At that time, the actions of sodium iodide, a chaotropic ion,and surfactant prevent the precipitation of components suchas proteins in the samples, resulting in selective precipitationof DNA and glycogen.

DNA Extractor® Kit Data

Recovery rate of added DNA (%)

Examples of using DNA Extractor® Kit
DNA addition/recovery
test

100

Dilution ratio: 112

1/10 1/102 1/103
3
4
5

1
6

1/10 1/102 1/103
789

λ/Hind III 10 to 1,000 pg waslabeled with 32P and added
to 100 μL of human serum.
DNA was extracted using thisPCR
kit and the recovery rate wasproduct
calculated.

80
60
40
20
10 100
50
250

500

1,000

This method

Phenol method

Detection by PCR
Serum containing hepatitis
B virus (HBV) was diluted instages. DNA was extractedusing this kit and the phenol
method. HBV-DNA wasamplified with PCR, and DNA
was detected using agarose
gel electrophoresis.
Lane 1: Marker DNA( φ X174/Hae III)

Amount of DNA added (pg)Reference

Ishizawa, M. et al., Nucleic Acids Res., 19, 5792 (1991).

03

Genomic DNA extraction from human whole blood sample
Extraction from cow/horse blood, cultured cells, and tissues is also possible

DNA Extractor® WB Kit

Human genomic gene analysis requires the preparationof high-molecular genomic DNA. Although many types ofgenomic DNA extraction methods are practiced at present,
most of them use organic solvents such as phenol. Its
toxicity and extraction procedures requiring time and effort
are the disadvantages.
This kit is primarily for the extraction of genomic DNA fromhuman whole blood. A high yield of DNA with high purity
can be obtained in a single tube in a short time without
using hazardous phenol and chloroform.

Wako Cat. No.

291-50502

50 tests

Operation time: 1.5 hours

Sample amount: 500 μL/test

Features□ A high yield rate of high-molecular DNA of over 90%□ The entire process is completed in a single tube in about
1.5 hours.
□ Organic solvents such as phenol and chloroform are notrequired.

□ Extraction from human whole blood as well as cow/horse
blood, cultured cells, and tissues is also possible.

Blood (0.5 mL)□ The operation is easier than the phenol/chloroform
extraction method and suitable for processing multiple
samples.

Lysis Solution 0.5mL
10,000 × g, 4℃, 20 seconds□ The entire procedure is performed in a single tube,
reducing genomic DNA damage by mechanical cutting inoperation.

Precipitate
Lysis Solution 1mL
×2

Package Size

10,000 × g, 4℃, 20 seconds
Precipitate
Enzyme Reaction Solution 0.2mL
Protease Solution 10μL

Kit Contents [50 tests (sample 500 μL)]:
1. Lysis solution. ..................................................................... 65 mL × 2

37℃, 1 hour
Completely dissolve the precipitate
by tapping during the operation.
Sodium Iodide Solution 0.3mL
Mix by inversion
Isopropanol 0.5mL

2. Enzyme reaction solution................................................. 10 mL × 1
3. Sodium iodide solution..................................................... 15 mL × 1
4. Washing solution (A)......................................................... 50 mL × 1
5. Washing solution (B)......................................................... 50 mL × 1

10,000 × g, room temperature, 10 minutes
Precipitate
Washing Solution (A) 1mL
10,000 × g, room temperature, 5 minutes

6. Protease............................................................................... 10 mg × 1

Principle of DNA extraction

Precipitate
Washing Solution (B) 1mL

i. Cell	 membrane and cytoplasm are destroyed by surfactant,isolating cell nuclei.

10,000 × g, room temperature, 5 minutes

ii. The	nuclear membrane and nuclear protein are decomposedby protease to expose DNA.

Precipitate
Dry under reduced pressure
TE buﬀer
Genomic DNA solution

iii.	Protein and lipids are made soluble by the action of sodium
iodide, and DNA is precipitated with isopropanol.

DNA Extractor® WB Kit Data
Examples of using DNA Extractor® WB Kit
M

1

2

3

4

M: Marker DNA (λ/Hind III)
Lane 1: Undigested human genomic DNA
Lane 2: Genomic DNA/BamH I
Lane 3: Genomic DNA/EcoR I
Lane 4: Genomic DNA/Hind III
Agarose gel electrophoresis patternobtained through the digestion of
genomic DNA derived from human whole
blood using this kit with various restriction
enzymes.

Reference

04

Wang, L. et al., Nucleic Acids Res., 22, 1774 (1994).

M

1

2

3

M: Marker DNA ( φ X174/Hinf I)
Lane 1: 262 bp
Lane 2: 345 bp
Lane 3: 408 bp
Electrophoresis was performed by
PCR-amplifying genomic DNA derived
from human whole blood using this kit.
Various size of human β-globin gene
was PCR amplified using 1 μg ofgenomic DNA as a template.

Genomic DNA extraction from cow/horse whole blood
Genomic DNA with high purity can be efficiently extracted from cow/horse whole blood by partially changing the standard protocol. Proteasetreatment is performed for 2 hours for horse blood, whereas washing
operation with lysis solution is repeated 4 times for cow blood, followed bytreatment with protease for 4 hours.
The operation time is about 3 hours for horse blood and 5 hours for cow
blood.
Blood samples used were added with EDTA·2Na or heparin as an
anticoagulant agent.
1

2

3

4

Electrophoretic image of genomic
DNA extracted from cow/horse
Lane 1: λ/Hind III
Lane 2: Horse blood (EDTA·2Na)
Lane 3: Horse blood (Heparin)
Lane 4: Cow blood (EDTA·2Na)

Operation procedure
Extraction from horse blood
Treatment with protease is performed for 2 hours until there is no nuclear
pellet.
Follow the standard protocol except for this operation.
Extraction from cow blood
This operation, which isolate cell nuclei and wash it by adding lysis solution,is repeated 4 to 5 times, because it is difficult to remove foreign substancessuch as hemoglobin only with 2 operations as described in the protocol.
The reference is until the red pellets become transparent.
Since pellets after centrifugation are very prone to peeling, sufficientattention is required in decantation.
Also, perform treatment with protease for about 4 hours.

Yield and purity of genomic DNA extracted from cow/horse
Sample3μL of 100 μL extracted DNA solution was
loaded on a 1% agarose gel and DNA
was separated by electrophoresis.

DNA yield (μg/mL blood)

A260/A280

Horse (EDTA·2Na)

56.62

1.91

Horse (Heparin)

48.34

1.88

Cow (EDTA·2Na)

22.04

1.86

Notes) Each horse blood sample was collected from different horses.

DNA extraction from tissues
Genomic DNA can be extracted from tissues by adding 2 to 3 simple operations to thestandard protocol. The operation is completed in about 2 hours.
Resulting DNA has little contamination with RNA or protein and can be used in restriction
enzyme treatment, PCR, Southern blotting analysis, fingerprinting, etc.
1

2

3

4

5

6

7

8

Protocol (DNA extraction from tissues)
50-100 mg of tissues
Lysis Solution 1mL
Homogenize
10,000 × g, 4℃, 20 seconds

Examples of rat gene detection by PCR
Rat GAPDH gene fragment (983 bp) was
amplified using DNA extracted from each rat
tissue as a template.
➡ 983bp

A band of 983 bp could be detected in all
DNAs extracted from each tissue.

Lane 1: Marker DNA( φ X174/Hae III)
Lane 2: Stomach
Lane 3: Intestine
Lane 4: Liver
Lane 5: Pancreas
Lane 6: Skeletal muscles
Lane 7: Negative control
Lane 8: Positive control

Precipitate
Lysis Solution 1mL
Twice

Microtube mixer, 30 seconds
10,000 × g, 4℃, 20 seconds

Precipitate
Enzyme Reaction Solution 200μL
RNase 20μg/mL
Incubate, 50℃, 10 minutes
Protease 10μL
Incubate, 50℃, 60 minutes
10,000 × g, room temperature, 5 minutes
SupernatantSodium Iodide Solution 0.3mL, Mix by inversion
Isopropanol 0.5mL, Mix by inversion
10,000 × g, room temperature, 10 minutes

Yield and purity of DNA extracted from each rat tissue using this kit
Tissues

DNA yield (μg/100 mg of tissues)

A260/A280

Stomach175

1.92

Intestine

396

1.96

Liver

286

1.95

Pancreas

624

1.95

Skeletal muscles

67

1.85

Despite of variations in DNA yield per unit of tissues, DNA with high purity could be extracted.

Precipitate

Washing Solution (A) 1mL, Mix
10,000 × g, room temperature, 5 minutes

Precipitate

Washing Solution (B) 1mL, Mix
10,000 × g, room temperature, 5 minutes

Precipitate
Dry under reduced pressure, 2 minutes
TE buﬀer 100-200 μL
DNA solution

05

Q&A for DNA Extractor® WB Kit

Q1. What is the principle of DNA Extractor® WB Kit?
This kit is for the extraction of genomic DNA from whole bloodand cultured cells. Only cell nuclei are collected by adding a lysis
solution containing surfactant first. Next, the nuclear membrane
and foreign substances are decomposed by protease to releaseDNA. Then, after protein is made soluble with sodium iodide,isopropanol is added to precipitate and collect genomic DNA
only. Also, with this method, the whole DNA extraction process
can be performed in a single tube.
Q2. Isn’t it contaminated with RNA?
Although contamination with RNA is rare in extraction fromwhole blood, cultured cells will be contaminated with RNA moreor less. In this case, RNA can be almost completely removed by
newly adding the treatment with RNase at 37°C for 10 minutes
to the basic protocol.
Q3. The yield rate of DNA is low. Why?
The followings are the possible reasons.
1. First centrifugation operation
For the centrifugation operation at 10,000 × g for 20 seconds,
perform centrifugation for 20 seconds after the centrifuge
completely reached 10,000 × g. Cell nuclei will not completely
detach in 20 seconds after the start button of the centrifuge ispressed; therefore, the DNA yield rate may drop by 20 to
30%. Although it varies by centrifuge, 10,000 × g is equivalent
to approximately 12,000 rpm in case of a tabletop centrifuge.
2. Supernatant removing operation after centrifugation
Perform all operations by decantation. Removal with a pipette
may result in a low yield of DNA.
3. DNA dissolution
As DNA in high concentration is extracted, add TE buffer(100-200 μL) and slowly dissolve it at room temperature for 3
hours or at 4°C overnight. DNA will not be completely dissolvedin a short time, which may result in an apparent low yield.
Q4. Can DNA be extracted from 100 μL of blood?
Yes. In this case, use the standard protocol by scaling it down.
Also, when the amount of blood is 100 μL or less, add physiological saline to make up to 100 μL and perform the sameprocedure. One kit can be used 250 tests for 100 μL of blood.
Q5. Can DNA be extracted by using heparin as an anticoagulant
agent instead of EDTA?
DNA can be successfully extracted at a usual concentration of
heparin (10 units/mL of blood) or less, but the yield rate markedly
decreases from a concentration of around 20 units/mL. Also,
DNA extracted from blood added with heparin may affectrestriction enzyme treatment or PCR reaction. Therefore, the useof EDTA·2Na or EDTA·2K (1 mg/mL of blood) is recommended.
Q6. Can DNA be extracted from stored blood?
DNA can be extracted successfully if blood is stored at 4°C for 3
weeks or less. However, in case of a long-term storage of 1
month or more, DNA will be gradually decomposed. Blood
should be stored frozen in case of long-term storage.
Q7. What should be done without a microtube mixer?
Mix the sample by inversion for about 1 to 2 minutes.
Q8. We want to suspend the experiment.
Store the precipitate separated by centrifugation with theaddition of lysis solution at -20°C.
Q9. We do not have a refrigerated centrifuge.
Extraction can be performed with a tabletop centrifuge. Inthat case, perform the operation quickly.

06

Q10. How much DNA can be extracted from 1 mL of blood?
Although there are variations by sample, approximately 20 to70 μg of DNA can be extracted from normal humans.
Q11. Can DNA be extracted from tissues with this kit?
Yes. DNA can be extracted by fragmenting tissues
by homogenization.
Q12. What is the initial cell number when extracting DNA from cultured
cells?
3
6
10 to 10 cells will be enough for extraction. Cultured cells will
be contaminated with RNA; therefore, before treatment withprote-ase, add 20 μg/mL of RNase A and treat at 37°C for 10
minutes.
Q13. Can mitochondrial DNA be extracted?
Yes. Please see the following publication.
Kubota, N., Hayashi, J., Inada, T. and Iwamura, Y.: Radiation
Research, 148, 395 (1997).
For the extraction of mitochondrial DNA alone, the use of
mtDNA Extractor® WB Kit (Wako Cat. No. 293-54401) and
mtDNA Extractor® CT Kit (Wako Cat. No. 291-55301) isrecommended.
Q14. What are the differences from DNA Extractor® Kit (Wako Cat. No.
295-50201)?
The purpose of use is different. DNA Extractor® Kit is for the
extraction of a slight amount of viral DNA in serum and a slightamount of host cell-derived DNA in biological products, while
DNA Extractor® WB Kit specifi-cally extracts genomic DNA fromwhole blood and cultured cells.
Q15. How should protease be stored after prepared?
It is stable at -20°C for 6 months.
Q16. What are the differences from DNA Extractor® WB-Rapid Kit?
DNA Extractor® WB-Rapid Kit is an improved product toenable DNA extraction in about 20 minutes. It is suitable fortreating many samples in a small volume.
Q17. Some insoluble matters still remain after treatment with proteaseat 37°C for 1 hour. What should be done?
Failure to digest in an hour is considered attributable toinsufficient washing with lysis solution in the first step. Whencell nuclei are precipitated in the beginning, repeat washing until
red pigment derived from hemoglobin is eliminated.
Q18. When DNA extraction from cultured cells was performed, white
insoluble matters emerged at the time of adding sodium iodide.
Should we move on to the next step after removing thesematters?
Insoluble matters may be observed depending on types ofcultured cells. In this case, there is no problem to move to thenext step, washing operation with washing solution (A), without
removing the matters. Insoluble matters will be dissolved byadding washing solution (A). If the purity of resulting DNA is low,
it is recommended to repeat the operation of cell nuclei
precipitation by lysis solution.
Q19. The A260/A280 ratio of DNA is 1.8 or below. What should be done?
Sodium iodide may be remained in DNA. In this case,repeat washing with washing solution (B).
Q20. Is there any reference literature?
See the following reference: Wang, L., Hirayasu, K., Ishizawa,
M. and Kobayashi, Y.: Nucl. Acids Res., 22, 1774 (1994).

Genomic DNA extraction from serum and plasma

DNA Extractor® SP Kit

Wako Cat. No.

296-60501

This kit, an improved version of DNA Extractor® Kit, is a
DNA extraction kit optimized for the extraction of DNA
fragments in serum or plasma. It can obtain a very highDNA yield, so is useful as a pretreatment reagent to detectand analyze the target gene with a high sensitivity. Also,
this product uses the sodium iodide method without usingorganic solvents such as phenol and chloroform.

Package Size

50 tests

Operation time: 1 hour or more
Sample amount: 100 μL/test

Features□ DNA can be obtained at a high yield rate from a small
amount (100 μL) of serum and plasma sample.

□ “Sodium iodide method” achieving nearly 100% yield rate
is adopted.
□ Organic solvents such as phenol and chloroform are notrequired.

Methods
100μL (200μL) *1□ No use of solid phase extraction method using silica
carrier realizes a high yield of DNA.

Serum or plasma *2□ High-quality DNA suitable for PCR can be obtained.

Enzyme Reaction Solution 200μL (300μL)□ Lipids, etc., derived from blood sample can be completely
removed with specially prepared alcohol solution includedin the kit.

Protein Digestion Solution 5μL (8～10μL)*3

Vortex
Incubate 56℃, 10 minutes (56℃, 30 minutes)
Sodium Iodide Solution 300μL (400μL)
Gently mix
Alcohol Solution 600μL (900μL)
VortexRoom temperature, 10 minutes□ The entire operation is completed in a single tube.

Kit Contents [50 tests (sample 100 μL)]:
1. Enzyme reaction solution................................................. 10 mL × 1

12,000 - 20,000×g, room temperature, 10 minutes
Precipitate

2. Protein digestion solution............................................... 250 μL × 1*4

3. Sodium iodide solution..................................................... 15 mL × 1

Washing Solution (A) 1mL (1.5mL) *5

4. Alcohol solution. ................................................................. 30 mL × 1

Vortex12,000 - 20,000×g, room temperature, 5 minutes*6

Precipitate

6. Washing solution (B)......................................................... 50 mL × 1*4

Washing Solution (B) 1mL (1.5mL)
Vortex *6
12,000 - 20,000×g, room temperature, 10 minutes
Precipitate

5. Washing solution (A)......................................................... 50 mL × 1

Principle of DNA extraction*4

Dry, 65℃, 5 minutes

TE buﬀer（10～20μL）
Vortex *6

Dissolve, 65℃, 3-5 minutes
Vortex *7
DNA solution Store at -20℃

i. Protein	
derived from serum/plasma is decomposed byprotease.
ii. 	Protein and lipids derived from serum/plasma are madesoluble by the action of sodium iodide and removed withalcohol solution included in the kit to precipitate DNA.

＊ 1 The protocol starting with 200 μL of sample is shown in the parentheses.
＊ 2	Handle serum (plasma) samples on the ice. Add enzyme reaction solution as early as
possible so that DNase in serum (plasma) will not be activated.
＊ 3 Take it out of the kit and transfer it on the ice before use.
＊ 4	Discard the supernatant, place the tube upside down on a paper towel, and remove
remaining supernatant by pressing the mouth of the tube to the paper towel.
＊ 5 After washing solution (A) is added, the sample can be stored for a long term at -20°C.
＊ 6 Mix well until the precipitate peels off from the wall of the tube.
＊ 7 Dissolve completely until there is no precipitate.
References

1) Ishizawa, M. et al., Nucleic Acids Res., 22, 1774 (1994).
2) Sozzi, G. et al., Clin. Cancer Res., 5, 2689 (1999).
3) Silva, J. M. et al., Cancer Res., 59, 3251 (1999).
4) Shao, Z. M. et al., Clin. Cancer Res., 7, 2222 (2001).

07

DNA Extractor® SP Kit Data
Examples of using DNA Extractor® SP Kit
Amplification of p53-Exon 5 region of DNA extracted from human serum and plasma
Using this kit, DNA was extracted from human serum (10 samples) and human plasma (1 sample) and dissolved in 20 μL of TE buffer (pH 8.0) to prepare
DNA samples. The p53-Exon 5 region (308 bp) was amplified from 5 μL of each sample.
The kit was compared with DNA extraction kit (Company A) which is based on the extraction principle with silica carrier (centrifugal filtration method) as
control.
Serum 1 Serum 2 Serum 3 Serum 4 Plasma Serum 5 Serum 6 Serum 7 Serum 8 Serum 9 Serum 10
M W

A

W

A

W

A

W

A W

A

W

A

W

A

W

A W

A

W

A

W

A

M: DNA Step Ladder Mix (80-10 kbp)
W: DNA Extractor® SP Kit
A: Kit of Company A➡ 308bp
PCR: 40 cycles
3% Agarose

Method of extracting DNA from 1 to 5 mL sample using DNA Extractor® SP Kit
After dissolving in TE buffer, insoluble matters were precipitated and absorption was detected in the background (absorbance 320 nm), indicating lower purity
than the standard method (extraction from 100- 200 μL). However, amplification by PCR can be performed successfully.
Method for treating 1 mL serum (plasma) sample
1 mL sample/15 mL centrifuge tube

7.5mL Enzyme Reaction Solution

50μL Protein Digestion Solution

250μL Protein Digestion Solution

56℃, 30 minutes

56℃, 30 minutes

3mL Sodium Iodide Solution

10mL Sodium Iodide Solution

6mL Alcohol Solution

22.5mL Alcohol Solution

Room temperature, 15 minutes

Room temperature, 15 minutes

10,000 rpm, 20 minutes

10,000 rpm, 20 minutes

38mL Washing Solution (A)10,000 rpm, 10 minutes

10,000 rpm, 10 minutes
Precipitate

10mL Washing Solution (B)38mL Washing Solution (B)

10,000 rpm, 10 minutes

10,000 rpm, 10 minutes

Precipitate(Dissolve in an appropriate amount of TE, etc.)

5mL

NC

M

308bp ➡

Precipitate

10mL Washing Solution (A)Precipitate

1mL

5 mL sample/50 mL centrifuge tube

2mL Enzyme Reaction Solution

Precipitate

08

Method for treating 5 mL serum (plasma) sample

Precipitate(Dissolve in an appropriate amount of TE, etc.)

NC: Negative Control
M: Marker

The p53-Exon 5 region (308 bp) samples forPCR (35 cycles). Of DNA extracted from 1 mL
and 5 mL of serum, DNA equivalent to 10 μL
of serum was applied to PCR. The target DNA
sequence was successfully amplified.

DNA extraction for 8-OHdG measurement
DNA extraction from human and animal parenchyma tissues

DNA Extractor® TIS Kit

Wako Cat. No.

296-67701

The sodium iodide method, the basic principle of DNA Extractor® Kit series,
is known as a DNA extraction method causing relatively little oxidation
during operations. This kit further inhibits oxidation of DNA with the use ofan oxidation inhibitor and is useful for the measurement of 8-OHdG(8-hydroxy-2'-deoxyguanosine), an oxidative stress marker.

Methods
Protocol 1

Protocol 2 ＊ 1

Approximately 150 mg of tissues

Approximately 150 mg of tissues

Lysis Solution 1mL

Lysis Solution 1mL

Homogenize

600 × g, 4℃, 10 minutes

Precipitate

Lysis Solution 1mL
600 × g, 4℃, 10 minutes

Precipitate

Lysis Solution 1mL×2

600 × g, 4℃, 10 minutes

Precipitate
RNase Solution 1μL

Protein Digestion Solution 15μL

Oxidation Inhibitor 3μL
Vortex37℃, 10 minutes

Oxidation Inhibitor 3μL
Vortex37℃, 1 hour (Vortex every 20 minutes)
10,000 × g, room temperature, 5 minutes
Supernatant/1.5 mL tube

4. Protein digestion solution..................750 μL× 1

5. Oxidation inhibitor..............................350 μL × 1

6. Sodium iodide solution...................... 15 mL × 1

7. Alcohol solution. .................................. 30 mL × 1
8. PEG solution........................................ 20 mL × 1

Sodium Iodide Solution 300μL
10,000 × g, room temperature, 10 minutes

Sodium Iodide Solution 300μL
Alcohol Solution 600μL

Precipitate

10,000 × g, room temperature, 10 minutes

70％EtOH 1mL
10,000 × g, room temperature,
5 minutes
Precipitate (nucleic acid)

Precipitate

Dissolve in DDW or TE buﬀer

3. RNase solution..................................... 50 μL × 1

Alcohol Solution 600μL

Supernatant/1.5 mL tube

Precipitate（DNA）□ Useful for the detection and measurement of oxidative stress markers.
	
Sodium iodide method causes little
oxidation of DNA during operation.
	The use of oxidation inhibitor further
inhibits oxidation of DNA.

2. Enzyme reaction solution.................. 15 mL × 1

Enzyme Reaction Solution ＊2 300μL

10,000 × g, room temperature,
5 minutes

Features1. Lysis solution. ...................................... 75 mL × 2

Enzyme Reaction Solution ＊2 300μL

70％EtOH 1mL

Sample amount: 100 μL/test

Kit Contents [50 tests (sample 100μL)] :×2

Precipitate

Protein Digestion Solution 15μL
37℃, 1 hour (Vortex every 20 minutes)
10,000 × g, room temperature, 5 minutes

Operation time: 2 hours or more□ Suitable for DNA extraction from humanand animal parenchyma tissues.

Homogenate/2 mL tube

600 × g, 4℃, 10 minutes

50 tests□ Organic solvents such as phenol andchloroform are not required.

Homogenize

Homogenate/2 mL tube

Package Size×2

×2

TE buﬀer 400 μL
RNase Solution 1μL
Oxidation Inhibitor 4μL
37℃, 30 minutes (Vortex every 10 minutes)
PEG solution 400 μL
10,000 × g, 4℃, 10 minutes
Precipitate
70％EtOH 1mL
10,000 × g, 4℃, 5 minutes
Precipitate (DNA)Dissolve in DDW or TE buﬀer

Protocol 1 is a simplified method where treatment with RNase is performed before treatment with protease.
Protocol 2 is a method to collect DNA by polyethylene glycol (PEG) precipitation method where protease treatment and RNase treatment are performed in different
processes to enhance the purity of DNA.
There is no difference in the amount of 8-OHdG between the two methods, and the levels of DNA oxidation inhibition are comparable.
＊ 1 DNA purity can be enhanced with this protocol.
＊ 2 If there is any deposit in enzyme reaction solution, completely dissolve the deposit by heating it to 37°C or returning it to room temperature and mix before use.

09

DNA Extractor® TIS Kit Data
DNA extraction from mouse liver and 8-OHdG measurement
DNA was extracted from mouse liver (the same mouse) using DNA Extractor® WB Kit [Wako Cat. No. 291-50502] and DNA Extractor® TIS Kit [Protocol 1].
Resulting DNA was treated with nuclease P1, the amount of 8-OHdG was measured by High Sensitive 8-OHdG Check [Wako Cat. No. 307-07921] and HPLC/
ECD, and two methods were compared.

Measurement by HighSensitive 8-OHdG
Check

Amount of 8-OHdG (pg/μg DNA)

2.0
1.8
1.6

The amount of 8-OHdG
generated during operation
could be reduced to half
as compared with DNA
Extractor® WB Kit.
As a result, the kit showedsuperior performance asa DNA extraction kit for
8-OHdG measurement.

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0

Measurement by HPLC/ECD
dG concentration(µmol/L)

8-OHdG/105 dG

DNA Extractor® WB Kit

1.41

687

0.21

DNA Extractor® TIS Kit

0.59

692

0.08

[HPLC conditions]
Column: Wakosil-II 5C18 RS φ 4.6 mm × 150 mm
Eluent: 50 mmol/L sodium acetate (pH 5.2), 9% MeOH
Flow rate: 1 mL/min
Absorbance: UV 260 nm
ECD electrolytic potential: +600 mV

DNA
Extractor®
TIS KitDNA
Extractor®
WB Kit8-OHdG concentration(nmol/L)The amount of 8-OHdG generated during operation can be reduced to half or less ascompared with DNA Extractor® WB Kit.
Also, the level of 8-OHdG in deoxyguanosine has been revealed to be very low,
showing superior performance as a DNA extraction kit for 8-OHdG measurement.

8-OHdG Assay
Preparation Reagent Set
When measuring 8-OHdG in tissues, extracted DNA fromtissues need to be hydrolyzed. This product is a reagent kit
including enzyme, buffer, etc., used in pretreatment of 8OHdG measurement.
Add a specified amount of each reagent to DNA samples formeasurement to promote reaction, thereby reducing variability in the nuclease treatment status (mononucleotide release)and realizing stable measurement of 8-OHdG.

292-67801

Manufacturer
Cat No.

Product name

Package Size

307-07921

KOG-HS10/E

High Sensitive
8-OHdG Check

96 tests301-06101

KOG-200S/E

New 8-OHdG
Check

1 set

50 tests

Kit Contents [50 tests]:
1. Acetic acid buffer.............................................................. 950 μL × 1

2. Nuclease P1.................................................................. 500 units × 1

3. Tris buffer............................................................................... 1 mL × 1
4. Alkaline phosphatase solution.......................................... 50 μL× 1

Methods
DNA sample 200μg/150μL DDW

This is an ELISA kit used to determine 8-OHdG amount, a biomarker of DNA
oxidative damage.
NIKKEN SEIL Co, Ltd.

Treatment by boiling 98℃, 2 minutes
Rapid cooling (on ice), 5-10 minutes
Acetic acid buﬀer 19 μL
Nuclease P1 solution 10 μL
37℃, 30 minutes
Tris buﬀer 20μL
Alkaline phosphatase solution 1 μL
37℃, 30 minutes
10,000 MW ultraﬁltration(spin ﬁltration 15,000 × g, 20 minutes)
Filtrate (DNA concentration: 1 mg/mL)
8-OHdG measurement(HPLC method or ELISA method)

10

Package Size

(pretreatment before 8-OHdG measurement)

Related productWako Cat. No.

Wako Cat. No.

DNA extraction from forensic samples(DNA extraction from trace amount of samples such as body hairs, blood stains, saliva stains, and nails)

DNA Extractor® FM Kit

This kit is to extract trace amount of DNA from forensic
samples such as body hairs (hairs), blood stains, saliva
stains, and nails used for personal identification.
Although a low yield of DNA becomes an obstacle indetermination in some cases, this kit rapidly dissolves
samples and efficiently collects DNA suitable for theamplification of STR (short tandem repeat) and hypervariable region of mitochondrial DNA. Also, this product uses
the sodium iodide method without using organic solvents
such as phenol and chloroform.

Protocol for DNA recovery from human hairs
Hair washed with ethanol (1 cm or less)
Lysis Solution 190μL
Enzyme-activated Reagent 10μL
Protease 10μL
Conﬁrm that hairs are completely soaked.
Incubate, 56-60℃, 20-40 minutes
9,600 - 21,600 × g, 1-2 minutes

Wako Cat. No.

Package Size295-58501

50 tests

Operation time: 41 minutes or more

Sample amount: hair 1 cm or less, nail 0.5 mg or less, stain
2 pieces of 5 mm × 5 mm

Features□ DNA can be extracted from low-soluble and trace amountsamples such as body hairs (hairs), blood stains, andnails.
□ DNA can also be extracted from hair shaft.

□ No solid-phase extraction reduces loss of trace amountDNA by adsorption to the carrier.

□ Organic solvents such as phenol and chloroform are notrequired.
□ DNA suitable for PCR can be obtained.

Kit Contents [50 tests]:
1. Lysis solution. .................................................................... 9.5 mL × 1
2. Enzyme activated reagent (EAR). ................................. 80 mg × 1
3. Reconstitution solution for EAR. ....................................500 μL× 1

4. Protease............................................................................... 10 mg × 1
Supernatant (200 μL)
Sodium Iodide Solution 250μL
Isopropanol 450 μL
13,800 - 21,600 × g, 4℃, 10 minutes
Precipitate
Washing Solution (A) 1mL
Mix
13,800 - 21,600 × g, 4℃, 5-10 minutes
Precipitate
Washing Solution (B) 1mL
Mix

5. Sodium iodide solution.................................................. 12.5 mL× 1

6. Washing solution (A)......................................................... 50 mL × 1

7. Washing solution (B)......................................................... 50 mL × 1

Sample volume◦ Body hair: 1 cm or less. Two hairs or less (2 cm or less)should be used if possible due to possible inhibition of PCR
reaction by melanin.
◦ Spots such as blood and saliva stains:
Cut into a size of about 5 mm × 5 mm.
Use up to 2 pieces of a 5 mm × 5 mm spot per test.
◦ Nail: Cut a fingernail of 0.5 mg or less (size of approximately
1 mm × 1 mm) into pieces of about 0.5 mm × 1 mm. DNA
extraction will become easier with smaller pieces.

13,800 - 21,600 × g, 4℃, 5-10 minutes
Precipitate
Dry by heating or reduced pressure
TE buﬀer 20-50 μL

DNA solution

Reference

Hair root

Hair shaft

Approximately
1 cm

2-6 cmTip of hairWang, L. et al., Nucleic Acids Res., 22, 1774 (1994).

11

DNA Extractor® FM Kit Data
Nucleic acid amplification reaction in hypervariable region of mitochondrial DNA (nucleotide positions
16159-16401) by using DNA extracted from hair root.
♀1

♀2

♀3

♂1

♂2

♂3

M

＋−

The lengths of hair shaft are all 0.5 cm, 1.0 cm, and 1.5 cm from the left ofthe lane for males and females.
The same band (240 bp) was detected in all subjects except for female 3.
Female 3 has dyed permed hair.
➡ 240bp

Amplification of hypervariable region of mitochondrial DNA by using DNA extracted from hair dyed withdifferent types of dyes.

M

Permed Manicured Colored
Henna
hair
hair
hair

Normal hair

♂

♀

＋−

The hair lengths are all 0.5 cm and 1.0 cm from the left of the lane.
The same band (240 bp) was detected in differently dyed hairs except for hairs dyed withhair color.
For hairs dyed with hair color, DNA itself in hairs may be affected by decomposition, etc.,
or the dye may have PCR inhibitory effect.
➡ 240bp

Description

Wako Cat. No.

Grade

Package Size

DNA Extractor Kit

295-50201

For genetic research

50 tests

DNA Extractor® WB Kit

291-50502

For genetic research

50 tests

DNA Extractor SP Kit

296-60501

For genetic research

50 tests

DNA Extractor® TIS Kit

296-67701

For genetic research

50 tests295-58501

For forensic medicine

50 tests®

®

®

DNA Extractor FM Kit

Wako Cat. No.

Grade/Manufacturer name

Package Size

8-OHdG Assay Preparation Reagent Set

Description

292-67801

For genetic research

50 tests

High Sensitive 8-OHdG Check

307-07921

NIKKEN SEIL Co, Ltd.

96 tests

New 8-OHdG Check

301-06101

NIKKEN SEIL Co, Ltd.

1 set

Listed products are intended for laboratory research use only, and not to be used for drug, food or human use. / Please visit our online catalog tosearch for other products from Wako; http://www.e-reagent.com/ This leaflet may contain products that cannot be exported to your country due toregulations. / Bulk quote requests for some products are welcomed. Please contact us.

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