Manual
Ver. 3.0

NIPPON GENE CO., LTD.

Contents

Ⅰ Description

2

Ⅱ Product Components

2

Ⅲ Storage Conditions

3

Ⅳ Directions

3

Ⅴ Protocol

3＜Rice DNA extraction protocol＞

4＜A grain of Rice DNA extraction protocol＞

5＜Rape seed DNA extraction protocol＞

6＜A grain of Rape seed DNA extraction protocol＞

7＜Potato DNA extraction protocol＞

8

Ⅵ Data Collection

10

1.

DNA extraction from Rice and Restriction Enzyme digestion

102.

Absorption spectrum of Rice DNA

103.

DNA extraction from a grain of Rice11

4.

PCR of DNA extracted from a grain of Rice12

5.

DNA extraction from Rape seed and Restriction Enzyme digestion

12

6.

Absorption spectrum of Rape seed DNA

13

7.

DNA extraction from a grain of Rape seed

13

8.

DNA extraction from Potato and Restriction Enzyme digestion

14

9.

Absorption spectrum of Potato DNA

14

10. PCR of DNA extracted from Potato

15

11. DNA extraction from other cereals

16

Ⅶ Troubleshooting

17

Ⅷ Related Products

18１

GM quicker 2 Manual Ver. 3.0

Ⅰ Description
GM quicker 2 is a kit to extract DNA from rice and other grains. This kit uses the principle
called Boom Technology that DNA is adsorbed by silica under the Chaotropicion existence.
Neither phenol nor chloroform is used in the extract operation.
The method of using DNA becomes widespread in the inspection of Genetically Modified
Organisms (GMO). Current DNA extraction kits were not necessarily efficient in the extraction
from grains, such as maize and soybean because their extractions object had been leaf.
In this kit, the object is focused on the grains so that high refinement level DNA can beextracted in only about 40 minutes. This kit is designed for not only nonglutinous rice but also
glutinous rice by using α-amylase, which enables the inspection of unknown sample of rice. It isalso possible to extract DNA for the inspection of a grain of rice or rape seed. The silica gel
membrane included in the spin column to be used with this kit has enough capacity to adsorb
sufficient DNA and enables high elution efficiency.
DNA obtained with GM quicker can be used for PCR and the restriction enzyme reaction.

Ⅱ Product Components
GE1 Buffer

40 ml × 1

GE2-K Buffer

5 ml × 1

GB3 Buffer

12.5 ml × 1

GW Buffer

40 ml × 1

TE （pH 8.0）

10 ml × 1

Proteinase K (20 mg/ml)

1 ml × 1

α-Amylase

0.1 ml × 1

RNase A (100 mg/ml)

0.5 ml × 1

Spin Column

50 sets

Manual1

Related Products: refer to p. 18.

２

GM quicker 2 Manual Ver. 3.0

Ⅲ Storage Conditions
Proteinase K and α-Amylase should be stored at -20°C. The remaining kit components can be
stored at room temperature (15-25°C). RNase A is stable for longer periods when stored at 4°C
or -20°C. GW Buffer includes ethanol, keep the buffer bottle tightly closed after use.

Ⅳ Directions• This kit should be used for research only and not used for medicine or any other purpose.
• Please do not handle this kit without basic knowledge of the reagent.
• Please handle this kit according to the manual.
• NIPPON GENE is not responsible for any troubles caused by handling different from themanual.

Ⅴ Protocol＜Reagents and machine necessary besides this kit＞
• Isopropanol• Pipette• Pipette-tip• 2 ml tube
• 1.5 ml tube
• Food mill
• Centrifuge• Vortex mixer• Pestle

３

GM quicker 2 Manual Ver. 3.0

＜Rice DNA extraction Protocol＞① Crush the rice by the food mill, etc. to prepare the sample of rice powder.
* Another method: Put 20 grains of rice into 2.0 ml tube, and add 700 μl of GE1
Buffer. Incubate at room temperature for 20 minutes. Crush the rice by a pestle. Add
20 μl of Proteinase K, 2 µl of α-Amylase, and 10 µl of RNase A. Mix by vortexing
for 30 seconds. Follow the procedure of ③.
② Weigh out 0.5 g of the rice powder, and transfer to a 2.0 ml tube. Add 700 μl of GE1 Buffer,
20 μl of Proteinase K, 2 μl of α-Amylase, and 10 μl of RNase A . Mix well by vortexing for
30 seconds.（*１）* It is not necessary to add α-Amylase if the rice sample does not include glutinous
rice obviously.
③ Incubate at 60℃ for 15 minutes.
* Incubate at 65℃ for 15 minutes if the sample does not include glutinous rice.
④ Add 85 μl of GE2-K Buffer,（*２） and mix well by vortexing.
⑤ Centrifuge at ≧13000 x g for 5 minutes at room temperature.（*３）⑥ Transfer 400 μl of supernatant to a fresh 1.5 ml tube.（*４）⑦ Add 150 μl of GB3 Buffer and then add 150 μl of isopropanol. Mix well by inverting 10-12
times.（*5）⑧ Transfer all the mixture made in ⑦ to Spin Column, and centrifuge at 13000 x g, for 30
seconds at room temperature. Discard the liquid in the collection tube.
⑨ Add 650 μl of GW Buffer to Spin Column, and centrifuge at 13000 x g for 60 seconds atroom temperature. Discard the liquid in the collection tube.
⑩ Remove the Spin Column to a fresh 1.5 ml tube.
⑪ Add 50 μl of TE (pH8.0) to the Spin Column, and incubate at room temperature for 3
minutes.
⑫ Centrifuge at 13000 x g for 60 seconds at room temperature, and collect the DNA solution.

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GM quicker 2 Manual Ver. 3.0

＜A grain of Rice DNA extraction protocol＞① Wrap a grain of rice in aluminum foil and crush it by a hammer, etc. to prepare the sampleof rice powder.
* Another method: Put a grain of rice into 1.5 ml tube and add 250 μl of GE1 Buffer.
Incubate at room temperature for 20 minutes. Crush the rice by a pestle. Add 10 μl ofProteinase K, 2 μl of α-Amylase, and 5 μl of RNase A. Mix by vortexing for 30
seconds. Follow the procedure of ③.
② Transfer the rice powder to a 1.5 ml tube. Add 250 μl of GE1 Buffer, 10 μl of Proteinase K,
2 μl of α-Amylase, and 5 μl of RNase A. Mix well by vortexing for 30 seconds.（*１）* It is not necessary to add α-Amylase if the rice sample does not include glutinousrice.
③ Incubate at 60℃ for 15 minutes.
* Incubate at 65℃ for 15 minutes if the sample does not include glutinous rice.
④ Add 40 μl of GE2-K Buffer,（*２） and mix well by vortexing.
⑤ Centrifuge at ≧13000 x g for 5 minutes at room temperature.（*３）⑥ Transfer 200 μl of supernatant to a fresh 1.5 ml tube.（*４）⑦ Add 75 μl of GB3 Buffer and then add 75 μl of isopropanol. Mix well by inverting 10-12
times.（*5）⑧ Transfer all the mixture made in ⑦ to Spin Column, and centrifuge at 13000 x g, for 30
seconds at room temperature. Discard the liquid in the collection tube.
⑨ Add 650 μl of GW Buffer to Spin Column, and centrifuge at 13000 x g for 60 seconds atroom temperature. Discard the liquid in the collection tube.
⑩ Remove the Spin Column to a fresh 1.5 ml tube.
⑪ Add 50 μl of TE (pH8.0) to the Spin Column, and incubate at room temperature for 3
minutes.
⑫ Centrifuge at 13000 x g for 60 seconds at room temperature, and collect the DNA solution.

５

GM quicker 2 Manual Ver. 3.0

＜Rape seed DNA extraction Protocol＞① Crush the rape seeds by the food mill, etc. to prepare the sample of rape seed powder.
② Weigh out 0.2 g of the rape seed powder, and transfer to 2.0 ml tube. Add 800 µl of GE1
Buffer, 20 µl of Proteinase K, and 10 µl of RNase A. Mix well by vortexing for 30 seconds.
（*１）③ Incubate at 65℃ for 15 minutes.
④ Add 100 µl of GE2-K Buffer,（*２） and mix well by vortexing.
⑤ Centrifuge at ≧13000 x g for 5 minutes at room temperature.（*３）⑥ Transfer 350 µl of supernatant to a fresh 1.5 ml tube.（*４）⑦ Add 130 µl of GB3 Buffer.
⑧ Add 130 µl of isopropanol and mix well by inverting 10-12 times.（*5）⑨ Transfer all the mixture made in ⑧ to Spin Column, and centrifuge at 13000 x g, for 30
seconds at room temperature. Discard the liquid in the collection tube.
⑩ Add 650 µl of GW Buffer to Spin Column, and centrifuge at 13000 x g for 60 seconds atroom temperature. Discard the liquid in the collection tube.
⑪ Remove the Spin Column to a fresh 1.5 ml tube.
⑫ Add 50 µl of TE (pH8.0) to the Spin Column, and incubate at room temperature for 3
minutes.
⑬ Centrifuge at 13000 x g for 60 seconds at room temperature, and collect the DNA solution.

６

GM quicker 2 Manual Ver. 3.0

＜A grain of Rape seed DNA extraction Protocol＞① Put a grain of rape seed into 1.5 ml tube, and crush it by a pestle.
② Add 250 μl of GE1 Buffer, 10 μl of Proteinase K, and 5 μl of RNase A. Mix well byvortexing for 30 seconds.（*１）③ Incubate at 65℃ for 15 minutes.
④ Add 40 μl of GE2-K Buffer,（*２） and mix well by vortexing.
⑤ Centrifuge at ≧13000 x g for 5 minutes at room temperature.（*３）⑥ Transfer 200 μl of supernatant to a fresh 1.5 ml tube.（*４）⑦ Add 75 μl of GB3 Buffer.
⑧ Add 75 μl of isopropanol and mix well by inverting 10-12 times.（*5）⑨ Transfer all the mixture made in ⑧ to Spin Column, and centrifuge at 13000 x g, for 30
seconds at room temperature. Discard the liquid in the collection tube.
⑩ Add 650 μl of GW Buffer to Spin Column, and centrifuge at 13000 x g for 60 seconds atroom temperature. Discard the liquid in the collection tube.
⑪ Remove the Spin Column to a fresh 1.5 ml tube.
⑫ Add 50 μl of TE (pH8.0) to the Spin Column, and incubate at room temperature for 3
minutes.
⑬ Centrifuge at 13000 x g for 60 seconds at room temperature, and collect the DNA solution.

７

GM quicker 2 Manual Ver. 3.0

＜Potato DNA extraction Protocol＞* When DNA is extracted from frozen dry potato, Proteinase K and α-Amylase would not be
added. Please follow the rice protocol (p. 4).
① Cut raw potato into 3 mm cubes by knife.
② Weigh out 0.3 g of the potato cut small, and transfer to a 1.5 ml tube. Add 500 µl of GE1
Buffer and 4 µl of RNase A(100 mg/ml). Crush well by a pestle.（*6）③ Mix well by vortexing for 30 seconds.
④ Add 85 µl of GE2-K Buffer,（*２） and mix well by vortexing.
⑤ Centrifuge at ≧13000 x g for 5 minutes at room temperature.（*3）⑥ Transfer 400 µl of supernatant to a fresh 1.5 ml tube.（*４）⑦ Add 150 μl of GB3 Buffer.
⑧ Add 150 µl of isopropanol, and mix well by inverting 10-12 times.（*5）⑨ Transfer all the mixture made in ⑧ to Spin Column, and centrifuge at 13000 x g, for 30
seconds at room temperature. Discard the liquid in the collection tube.
⑩ Add 600 μl of GW Buffer to Spin Column, and centrifuge at 13000 x g for 60 seconds atroom temperature. Discard the liquid in the collection tube.
⑪ Remove the Spin Column to a fresh 1.5 ml tube.
⑫ Add 50 μl of TE (pH8.0) to the Spin Column, and incubate at room temperature for 3
minutes.
⑬ Centrifuge at 13000 x g for 60 seconds at room temperature, and collect the DNA solution.

８

GM quicker 2 Manual Ver. 3.0

(*1)

If mixing is insufficient, DNA yield is decreased remarkably. If the 2.0 ml or 1.5 ml
tube is not vertical, the mixture becomes foamy, which decreases mixing efficiency.
The tube should be vertical to the vortex mixer and mix well for 30 seconds. If mixing
is insufficient, mix for 30-60 another seconds.

(*2)

Please add GE2-K Buffer even though the foam generated by ② remains. Because theviscosity of the mixture has risen, please invert carefully until GE2-K Buffer is mixed
well.

(*3)

Please confirm the maximum speed of centrifuge rotor and the maximum centrifugation
stability (x g) of 50 ml tube.

(*4)

Please avoid taking neither precipitate nor float.

(*5)

Add GB3 Buffer before ethanol or isopropanol, and then mix. If the precipitation
appears, mix well by inverting until the liquid becomes clear.

(*6)

Crush the potato sample after adding GE1 Buffer. If the sample were crushed by foodmill before adding GE1 Buffer, a band of extracted genomic DNA by agarose gelelectrophoresis would be smear.

９

GM quicker 2 Manual Ver. 3.0

Ⅵ Data Collection
1. DNA extraction from Rice and Restriction Enzyme digestion
DNA could be extracted from rice with this kit. And DNA was digested by EcoRI.
1

2

3

4

5

6

7

8
Lane 1, 8：OneSTEP Marker 6(λ/ StyI digest)
Lane 2 ：Koshihikari genomic DNA intact
Lane 3 ：Koshihikari genomic DNA/ EcoRI digest
Lane 4 ：Thai rice genomic DNA intact
Lane 5 ：Thai rice genomic DNA/ EcoRI digest
Lane 6 ：Glutinous rice genomic DNA intact
Lane 7 ：Glutinous rice genomic DNA/ EcoRI digest＊400 ng of the extracted DNA was run
on a 1% Agarose S gel.

2. Absorption spectrum of Rice DNA
The absorption peak in the vicinity of A260 shows that DNA extracted with this kit is high purity.

0.35
0.30
0.25

Abs

0.20
0.15
0.10
0.05
0.00
220

240

260

280

300

320

Wavelength[ nm ]Fig.1 UV scan of Koshihikari rice DNA extracted with this kit.

１０

GM quicker 2 Manual Ver. 3.0

0.45
0.40
0.35
0.30

Abs

0.25
0.20
0.15
0.10
0.05
0.00
220

240

260

280

300

320

Wavelength[ nm ]Fig.2

UV scan of Thai rice DNA extracted with this kit.

0.35
0.30
0.25

Abs

0.20
0.15
0.10
0.05
0.00
220

240

260

280

300

320

Wavelength[ nm ]Fig.3 UV scan of Glutinous rice DNA extracted with this kit.

3. DNA extraction from a grain of RiceDNA was extracted from a grain of rice.
1

2

3

4

5

Lane 1, 5：OneSTEP Marker 6 ( λ / StyI digest)
Lane 2：Koshihikari unpolished rice genomic DNA

Lane 3：Koshihikari polished rice genomic DNA

Lane 4：Koshihikari boiled rice genomic DNA＊20 µl of the extracted DNA 50 μl was run
on a 1% Agarose S gel.

１１

GM quicker 2 Manual Ver. 3.0

4. PCR of DNA extraction from a grain of Rice
PCR could be performed using 0.1 µl of the DNA extracted from a grain of rice as a template
without any blocking.
1

2

3

5

4

6
Lane 1, 6：OneSTEP Marker 4 (φX174/ HaeIII digest)
Lane 2：Template was Koshihikari unpolished rice DNA.

Lane 3：Template was Koshihikari polished rice DNA.

Lane 4：Template was Koshihikari boiled rice DNA.

Lane 5：No Template Control＊10 µl of PCR products was run on a 2% Agarose S gel.

5. DNA extraction from Rape seed and Restriction Enzyme digestion
DNA could be extracted from rape seed with this kit. And DNA was digested by EcoRI.
1

2

3

4

5

6

7

8
Lane 1, 8 ：OneSTEP Marker 6(λ/ StyI digest)
Lane 2, 4, 6：Rape seed genomic DNA intact
Lane 3, 5, 7：Rape seed genomic DNA/ EcoRI digest＊400 ng of the extracted DNA was run
on a 1% Agarose S gel.

１２

GM quicker 2 Manual Ver. 3.0

6. Absorption spectrum of Rape seed DNA
The absorption peak in the vicinity of A260 shows that DNA extracted with this kit is high purity.

0.50
0.45
0.40
0.35

Abs

0.30
0.25
0.20
0.15
0.10
0.05
0.00
220

240

260

280

300

320

Wavelength[ nm ]Fig.4 UV scan of Rape seed DNA extracted with this kit.

7. DNA extraction from a grain of Rape seed
DNA was extracted from a grain of Rape seed.
1

2

3

4

5
Lane 1, 5 ：OneSTEP Marker6 (λ/ StyI digest)
Lane 2, 3, 4：Rape seed genomic DNA＊15 µl of the extracted DNA 50 µl was run
on a 1% Agarose S gel.

１３

GM quicker 2 Manual Ver. 3.0

8. DNA extraction from Potato and Restriction Enzyme digestion
DNA could be extracted from raw Potato with this kit. And DNA was digested by EcoRI.
1

2

3

5

4

6

7

8
Lane 1, 8：OneSTEP Marker 6 (λ/ StyI digest)
Lane 2 ：Danshaku genomic DNA intact
Lane 3 ：Danshaku genomic DNA/ EcoRI digest
Lane 4 ：May Queen genomic DNA intact
Lane 5 ：May Queen genomic DNA/ EcoRI digest
Lane 6 ：Frozen dry potato genomic DNA intact
Lane 7 ：Frozen dry potato genomic DNA/EcoRI digest＊200 ng of the extracted DNA was run
on a 1% Agarose S gel.

9. Absorption spectrum of Potato DNA
The absorption peak in the vicinity of A260 shows that DNA extracted with this kit is high purity.
0.4
0.35
0.3
Abs

0.25
0.2
0.15
0.1
0.05
0
220

240

260

280

300

320

Wavelength [ nm ]Fig.5 UV scan of May Queen potato DNA extracted with this kit.

１４

GM quicker 2 Manual Ver. 3.0

0.4
0.35
0.3
Abs

0.25
0.2
0.15
0.1
0.05
0
220

240

260

280

300

320

Wavelength [ nm ]Fig.6 UV scan of Danshaku potato DNA extracted with this kit.

10. PCR of DNA extracted from Potato
PCR could be performed using 25 ng of the DNA extracted from potato as a template fordetection of UGP gene without any blocking.
1

2

3

4

5

6

7
Lane 1,7： OneSTEP Marker 11 (φX174/ HaeIII digest)
Lane 2 ： Positive Control
Lane 3 ： No template Control
Lane 4 ： Template was Danshaku potato DNA.
Lane 5 ： Template was May Queen potato DNA.
Lane 6 ： Template was frozen dry potato DNA.

＊ 5 µl of PCR products was run

on a 3% Agarose 21 gel.
＊The amplicons were detected clearly.
＊Positive Control ： GM Potato Positive Control Plasmid (for qualitative analysis)
Code No. 317-06501

１５GM quicker 2 Manual Ver. 3.0

11. DNA extraction from other cereals
DNA was extracted from following cereals.

1

2

3

4

5

6

7

8

9

10

11

Lane 1, 11：OneSTEP Marker 6(λ/ StyI digest)
Lane 2： Soy bean genomic DNA
Lane 3： Maize genomic DNA
Lane 4： Wheat genomic DNA
Lane 5： Japanese millet genomicDNA
Lane 6： Millet genomic DNA
Lane 7： Foxtail millet genomicDNA
Lane 8： Hatomugi genomic DNA
Lane 9： Katamaru wheat genomicDNA
Lane 10：Amaranths genomic DNA＊ 400 ng of the extracted DNA was run on a 1% Agarose S gel.
＊ The amount of powder sample for extraction.
Soy bean ：50 mg
Maize, Wheat, Katamaru wheat, and Amaranths ：200 mg
Japanese millet, Millet, Foxtail millet, and Hatomugi ：300 mg

１６

GM quicker 2 Manual Ver. 3.0

Ⅶ Troubleshooting
TroubleLow DNA yield

Cause

Suggestion

Insufficient crush

Please crush the samples as in pieces as possible. It
is desirable that the sizes of the samples are wellmatched, so please pass the samples through a sieve
if necessary.

Extraction

Please mix well by vortexing after adding GE1

efficiency is low.

Buffer and RNase A.
If the 2.0 ml or 1.5 ml tube is not vertical, themixture becomes foamy, and mixing efficiency
would be decreased. The 50 ml tube should bevertical to the vortex mixer and mix well.

Insufficient

If the centrifugation for elution was performed

elution

immediately after adding TE to the Spin Column,
DNA yield would be low. Please incubate for
3minutes at room temperature.

Centrifugation at Please centrifuge at room temperature (25-15℃).
below 15℃
Incubate at above Please incubate at temperature described in the
70℃ after adding manual.
α-AmylaseandProteinase K.
RNA in the eluate

No activation of RNase A cannot be stored after it is mixed with GE1
RNase

Buffer. Please store them separately.

A large amount of Insufficient mix

Mix well after adding GE2-K Buffer.

white

precipitate(The white precipitate would remain in thethat appeared after

extraction. In the cases, mix well after adding

adding GB3 Buffer

GE2-K Buffer, and then centrifuge the mixture. The

remainssupernatant can be transferred to the Spin Column.)

evenisopropanoliforethanol is added.
Low OD260/280

Taking precipitate Please careful not to touch the precipitant by the tipor float to the when the supernatant is collected.
Spin Column.

１７

GM quicker 2 Manual Ver. 3.0

Ⅷ Related Products
Code No.

Product Name

Size

317-06361

GM quicker

50 reactions

311-07241

GM quicker 3

50 reactions

319-07161

GM quicker 96

96well-plate×4

317-07341

On-Site Column Set for GM quicker

20 reactions

314-06371

GE1 Buffer

500 ml

311-06381

GE2 Buffer

200 ml

318-06391

RNase A（100 mg/ml）

0.5 ml×5

311-06641

GW Buffer

40 ml×2

318-06651

GE2-K Buffer

100 ml

315-06661

GB3 Buffer

12.5 ml×2

312-06671

GM quicker 2 Enzyme Set

1 Set(Proteinase K 2 ml, α-Amylase 0.2 ml)
316-90025

TE (pH8.0)

500 ml318-90105

Distilled Water, Deionized, Sterile

500 ml

312-01193

Agarose S

100 g

313-03242

Agarose 21

25 g

312-06512

Agarose XP

25 g

310-06513

Agarose XP

100 g

311-02682

Agarose X

25 g

311-05281

OneSTEP Marker 6

1,500 μl

318-05791

OneSTEP Marker 4

375 μl

312-05831

OneSTEP Marker 11

375 μl

319-08141

Collection Tube

50 tubes×2

１８

GM quicker 2 Manual Ver. 3.0

NIPPON GENE CO., LTD.

If you have any questions, please contact us by web form.

http://www.nippongene.com/