Manual
Ver. 4.0

GM quicker は「組換え DNA 技術応用食品の検査方法」に準拠した試薬です。
(食安発第 0629002 号)

NIPPON GENE CO., LTD.

ContentsⅠ Description

2

Ⅱ Product Components

2Ⅲ Storage Conditions

3

Ⅳ Directions

3Ⅴ Protocol

3＜Maize DNA extraction protocol＞

4＜100 mg of Maize DNA extraction protocol＞

5＜Soybean DNA extraction protocol＞

6＜100 mg of Soybean DNA extraction protocol＞

7

Ⅵ Data Collection

9

１．DNA extraction from Maize and Soybean

9２．Absorption spectrum of Maize and Soybean DNA

9３．Restriction Enzyme digestion of Maize and Soybean DNA

10４．Detection of Maize Starch Synthase IIb and Soybean Lectin genes by PCR

10Ⅶ Troubleshooting

11

Ⅷ Related Products

12Ⅸ Simple protocol

13

１

GM quicker Manual Ver. 4.0

Ⅰ Description
GM quicker is a kit to extract DNA from grains, such as maize and soybean. This kit uses theprinciple 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, suchas 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 be extracted inonly about 35 minutes. This kit is designed considering operation problems such as the crossing
contaminations of the samples for the inspection. The silicagel 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

100 ml × 3

GE2 Buffer

37.5 ml × 1

GB3 Buffer

12.5 ml × 1

GW Buffer

40 ml × 1TE（pH8.0）

10 ml × 1

RNase A(100 mg/ml)

0.5 ml × 2

Spin Column

50 sets

Manual1＊ DNA extraction can be done 50 times for the sample 1g of the maize powder.
If the extraction should be done 50 times for the sample 1g of the soybean powder, GE1 Buffer,
GE2 Buffer, and RNase A would be insufficient. Please purchase them separately.
Related Products314-06371

GE1 Buffer

500 ml

311-06381

GE2 Buffer

200 ml

318-06391

RNase A（100 mg/ml）

0.5 ml×5２

GM quicker Manual Ver. 4.0

Ⅲ Storage Conditions
All the 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 the manual.

Ⅴ Protocol＜Reagents and machine necessary beside this kit＞
• Ethanol• Isopropanol• Ice• Pipette• Pipette-tip• 50 ml tube
• 2 ml tube
• 1.5 ml tube
• Food mill
• Centrifuge• Vortex mixer

３

GM quicker Manual Ver. 4.0

＜Maize DNA extraction Protocol＞① Crush the maize seeds by the food mill, etc. to prepare the sample of maize powder② Weigh out 1.0 g of the maize powder, and transfer to a 50 ml tube. Add 6 ml of GE1 Bufferand 20 μl of RNase A. Mix well by vortexing for 30 seconds（※１）③ Incubate at room temperature for 10 minutes.
④ Add 750 μl of GE2 Buffer,（※２）and mix well by inverting 10-12 times.
⑤ Chill on ice for 10 minutes.
⑥ Centrifuge at ≧5,000 x g for 10 minutes at 4℃.（※３）⑦ Transfer 4 ml of supernatant to a fresh 50 ml tube.（※４）⑧ Transfer 400 μl of supernatant from the 50 ml tube to a fresh 1.5 ml tube. Store the rest ofsupernatant at 4℃.（※５）⑨ Add 50μl of GB3 Buffer.
⑩ Add 200 μl of ethanol, and mix well by inverting 10-12 times.（※６）⑪ Move all the mixture made in ⑩ to Spin Column, and centrifuge at ≧13,000 x g, for 30
seconds at 4℃. Discard the liquid in the collection tube.
⑫ Add 600 μl of GW Buffer to Spin Column, and centrifuge at ≧13,000 x g for 60 seconds at
4℃. Discard the liquid in the collection tube.
⑬ Remove the Spin Column to a fresh 1.5 ml tube.
⑭ Add 50 μl of TE (pH8.0) to the Spin Column, and incubate at room temperature for 3 minutes.
⑮ Centrifuge at ≧13,000 x g for 60 seconds at 4℃, and collect the DNA solution.

４GM quicker Manual Ver. 4.0

＜100 mg of Maize DNA extraction protocol＞① Crush the maize seeds by the food mill, etc. to prepare the sample of maize powder② Weigh out 100 mg of the maize powder, and transfer to a 2 ml tube. Add 600μl of GE1 Buffer
and 4 μl of RNase A. Mix well by vortexing for 30 seconds（※１）③ Incubate at room temperature for 5 minutes.
④ Add 75 μl of GE2 Buffer,（※２）and mix well by inverting 10-12 times.
⑤ Chill on ice for 5 minutes.
⑥ Centrifuge at ≧13,000 x g for 5 minutes at 4℃.（※３）⑦ Transfer 400 μl of supernatant from the 50 ml tube to a fresh 1.5 ml tube.（※４）⑧ Add 50 μl of GB3 Buffer.
⑨ Add 200 μl of ethanol, and mix well by inverting 10-12 times.（※６）⑩ Move all the mixture made in ⑨ to Spin Column, and centrifuge at ≧13,000 x g, for 30
seconds at 4℃. Discard the liquid in the collection tube.
⑪ Add 600 μl of GW Buffer to Spin Column, and centrifuge at ≧13,000 x g for 60 seconds at4℃. Discard the liquid in the collection tube.
⑫ Remove the Spin Column to a fresh 1.5 ml tube.
⑬ Add 50 μl of TE(pH8.0) to the Spin Column, and incubate at room temperature for 3 minutes.
⑭ Centrifuge at ≧13,000 x g for 60 seconds at 4℃, and collect the DNA solution.

５GM quicker Manual Ver. 4.0

＜Soybean DNA extraction protocol＞① Crush the soybeans by the food mill, etc. to prepare the sample of soybean powder② Weigh out 1.0 g of the soybean powder, and transfer to a 50 ml tube. Add 12 ml of GE1 Buffer and40 μl of RNase A. Mix well by vortexing for 30 seconds.（※１）③ Incubate at room temperature for 10 minutes.
④ Add 1.5 ml of GE2 Buffer,（※２）and mix well by inverting 10-12 times.
⑤ Chill on ice for 10 minutes.
⑥ Centrifuge at ≧5,000 x g for 10 minutes at 4℃.（※３）⑦ Transfer 8 ml of supernatant to a fresh 50 ml tube.（※４）⑧ Transfer 700 μl of supernatant from the 50 ml tube to a fresh 2 ml tube. Store the rest ofsupernatant at 4℃.（※５）⑨ Add 250 μl of GB3 Buffer.
⑩ Add 250 μl of isopropanol, and mix well by inverting 10-12 times.（※６）⑪ Move 600 μl of the mixture made in ⑩ to Spin Column, and centrifuge at ≧13,000 x g, for 30
seconds at 4℃. Discard the liquid in the collection tube.
⑫ Add the remaining mixture made in ⑩ to the same Spin Column, and centrifuge at ≧13,000 x g,for 30 seconds at 4℃. Discard the liquid in the collection tube.
⑬ Add 600 μl of GW Buffer to the Spin Column, and centrifuge at ≧13,000 x g for 60 seconds at
4℃. Discard the liquid in the collection tube.
⑭ Remove the Spin Column to a fresh 1.5 ml tube.
⑮ Add 50 μl of TE(pH8.0) to the Spin Column, and incubate at room temperature for 3 minutes.
⑯ Centrifuge at ≧13,000 x g for 60 seconds at 4℃, and collect the DNA solution.

６GM quicker Manual Ver. 4.0

＜100 mg of Soybean DNA extraction protocol＞① Crush the soybeans by the food mill, etc. to prepare the sample of soybean powder② Weigh out 100 mg of the soybean powder, and transfer to a 2 ml tube. Add 600 μl of GE1 Buffer
and 4 μl of RNase A. Mix well by vortexing for 30 seconds（※１）③ Incubate at room temperature for 5 minutes.
④ Add 75 μl of GE2 Buffer,（※２）and mix well by inverting 10-12 times.
⑤ Chill on ice for 5 minutes.
⑥ Centrifuge at ≧13,000 x g for 5 minutes at 4℃.（※３）⑦ Transfer 400 μl of supernatant from the 50 ml tube to a fresh 1.5 ml tube.（※４）⑧ Add 150 μl of GB3 Buffer.
⑨ Add 150 μl of isopropanol, and mix well by inverting 10-12 times.（※６）⑩ Move all the mixture made in ⑨ to Spin Column, and centrifuge at ≧13,000 x g, for 30 seconds
at 4℃. Discard the liquid in the collection tube.
⑪ Add 600 μl of GW Buffer to Spin Column, and centrifuge at ≧13,000 x g for 60 seconds at 4℃.
Discard the liquid in the collection tube.
⑫ Remove the Spin Column to a fresh 1.5 ml tube.
⑬ Add 50 μl of TE(pH8.0) to the Spin Column, and incubate at room temperature for 3 minutes.
⑭ Centrifuge at ≧13,000 x g for 60 seconds at 4℃, and collect the DNA solution.

７

GM quicker Manual Ver. 4.0

※1If mixing is insufficient, DNA yield is decreased remarkably. If the 50 ml tube is notvertical, the mixture becomes foamy, which decreases mixing efficiency. The 50 ml tube
should be vertical to the vortex mixer and mix well for 30 seconds. If mixing isinsufficient, mix for 30-60 another seconds.

※2

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

※3

Please confirm the maximum speed of centrifuge rotor and the maximum centrifugation
stability (x g) of 50 ml tube. If the 50 ml tube is centrifuged at its maximum g, anadapter should be used and please confirm that the tube is not damaged by preliminary
examination.

※4Please avoid taking neither precipitate nor float.

※5

The rest of the supernatant can be used within 24 hours. However, white precipitation
would appear in the supernatant stored at 4℃. In this case, please mix by vortexing atroom temperature to dissolve before the operation is started.

※6

Add GB3 Buffer before isopropanol, and then mix. If the precipitation appears, mix wellby inverting until the liquid becomes clear.

８GM quicker Manual Ver. 4.0

Ⅵ Data Collection
1. DNA extraction from Maize and Soybean
DNA could be extracted from both of the seeds of Maize and Soybean with this kit.
12

3

45

6

78
Lane 1, 8 ：OneSTEP Marker 6(λ/ StyI digest)
Lane 2～4 ：Maize genomic DNA
Lane 5～7 ：Soybean genomic DNA＊ 1/25 of the extracted DNA was run on a 1%
Agarose S gel.

2. Absorption spectrum of Maize and Soybean DNA
The absorption peak in the vicinity of A260 shows that DNA extracted with this kit is high purity.
0.3
0.25

Abs

0.2
0.15
0.1
0.05
0220

240260280300320

Wavelength [ nm ]Fig.1

UV scan of Maize DNA extracted with this kit.
0.25

Abs

0.2
0.15
0.1
0.05
0220

240

260

280300320

Wavelength [ nm ]Fig.2

UV scan of Soybean DNA extracted with this kit.

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GM quicker Manual Ver. 4.0

3. Restriction enzyme digestion of Maize and Soybean DNA
DNA extracted with this kit was digested by EcoRI and HindIII.
12345678
Lane 1. 8：OneSTEP Marker6 (λ/ StyI digest)
Lane 2：Maize genomic DNA intact

Lane 3：Maize genomic DNA digest / EcoRI

Lane 4：Maize genomic DNA digest / HindIII

Lane 5：Soybean genomic DNA intact

Lane 6：Soybean genomic DNA / EcoRI

Lane 7：Soybean genomic DNA / HindIII

4. Detection of Maize Starch SynthaseⅡb (SSIIb) and Soybean Lectin (LeI) genes by PCR
PCR was performed using primers for detection of SSIIb and LeI.The PCR condition followed theJapanese Ministry of Agriculture, Forestry and Fisheries guideline, “Japanese Agricultural Standard(JAS) analysis manuals”.
123

45678
Lane 1, 8 ：OneSTEP Marker 11(pUC19/MspI digest)
Lane 2～4：Maize SSIIb (114 bp)
Lane 5～7：Soybean Le1 (118 bp)＊5μl of PCR products were run on a 3% Agarose 21 gel.

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GM quicker Manual Ver. 4.0

Ⅶ Troubleshooting
TroubleCause

Suggestion

Low DNA yield

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 ifnecessary.

Extraction

Please mix well by vortexing after adding GE1 Buffer

efficiency is low.

and RNase A. Especially, in the case for soybean, it iseasy to become lumpy, and the surface of the powder
does not come in contact with extraction buffer.
If the 50 ml tube is not vertical, the mixture becomesfoamy, and mixing efficiency would be decreased. The
50ml tube should be vertical to the vortex mixer andmix well.

Insufficient elution

If the centrifugation for elution were performed
immediately after adding TE to the Spin Column, DNA
yield would be low. Please incubate for 3 minutes atroom temperature.

RNA in the elute
A large amount of

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

RNase

Buffer. Please store them separately.

Insufficient icing

Please enlarge the contact area of ice and 50 ml tube towhite precipitate thatraise the cooling efficiency. I the case for soybean, mix

appeared after adding

well because the mixture becomes viscous after adding

GB3 Buffer remains

GE2 Buffer.

even if isopropanol or(The white precipitate would remain in the extraction

ethanol is added.

from other grains except maize and soybean. In thecases, mix well after adding GE2 Buffer, and thencentrifuge the mixture. The supernatant can betransferred to the Spin Column.)

Low OD260/280

Taking precipitate

The oil membrane might appear to the liquid level foror float to the Spinmaize after centrifugation in 50 ml tube. Inserting 1 mlColumn.

tip vertically in the liquid level and then lifting it
slowly could remove the oil material.
Please careful not to touch the precipitant by the tip
when the supernatant is collected.

１１

GM quicker Manual Ver. 4.0

Ⅷ Related Products
Code No.

Product Name

Size

310-06591

GM quicker 250 reactions

311-07241

GM quicker 350 reactions

316-07791

GM quicker 450 reactions

319-07161

GM quicker 96

96well-plate×4317-07341

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×2312-06671

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

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

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GM quicker Manual Ver. 4.0

Ⅸ Simple protocol

１３

GM quicker Manual Ver. 4.0

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GM quicker Manual Ver. 4.0

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GM quicker Manual Ver. 4.0１６

GM quicker Manual Ver. 4.0

＜ Note ＞

１７

GM quicker Manual Ver. 4.0

＜ Note ＞

１８

GM quicker Manual Ver. 4.0

NIPPON GENE CO., LTD.
If you have any questions, please contact us by web form.

http://www.nippongene.com/