ManualVer. 3.0

NIPPON GENE CO., LTD.

Contents

I Description

2

II Product Components

2

III Storage Conditions

2

IV Precautions

3

V Protocol selection related to JAS law display items

3

VI Protocol

4＜Protocol 1: DNA extraction method from dried foods＞

5＜Protocol 2: DNA extraction method from foods containing large amount of water＞
6
VII Data Collection

8

VIII Troubleshooting

12

IX Related Products

13

X Reference14１

GM quicker 4 Manual Ver. 3.0

I Description
GM quicker 4 is a kit for extracting and purifying DNA in food products. Since this kit applies
the principle that DNA is adsorbed to silica in the presence of chaotropic ion, DNA contained in foodproducts can be extracted without using toxic organic solvents such as phenol and chloroform.
The spin column used in this kit ensures the maximum column volume within the range in which
the rotor of a common centrifuge can be used, and the enclosed silica gel membrane ensures a highadsorption volume to recover DNA in food products.
Since DNA obtained by this kit can be used for the enzyme reactions such as PCR and LAMP, theapplication is expected to examine genetically-modified food products, specific raw materials, foodmicroorganisms, raw materials and the like.

II Product Components
GE1 Buffer

100 ml × 2 *GE2-M Buffer

20 ml × 1

GB3 Buffer

12.5 ml × 3 *GW Buffer

40 ml × 1 *TE (pH8.0)

10 ml × 1 *Proteinase K (20 mg/ml)

1 ml × 1 *α-Amylase (high concentration) 0.1 ml × 1 *RNase A (100 mg/ml)

0.5 ml × 1 *Spin Column

50 sets

Manual1* Each item can be purchased separately.

Please see page 13 for more details.

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.

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GM quicker 4 Manual Ver. 3.0

IV Precautions
・This product is a reagent for research and cannot be used for medicinal or other purposes.
・This product should be handled only by persons having a basic knowledge about reagents.
・Handle this product in accordance with the descriptions in the manual.
・ We are not responsible for any problems that may arise if product is not handled in accordancewith the manual.

V Protocol selection related to JAS* law display items (Reference)* Japanese Agricultural Standards (JAS)Protocol 1: Dried food products (freeze-dried food and the like)(1)Frozen and dried bean curd, okara (residue of soybean after soy milk isextracted) and yuba (dried bean curd film)(2) soybean powder

soybean(6) corn starch(4) dried potato(5) corn snack(3) broiled(7) popcorn(8)

potato snacks (9) potato starchProtocol 2: Food products having a high water content (liquid food products and food products
homogenized by adding sterilized water)(10) bean curd and fried bean curd (11) natto (fermented soybean) (12) soy milk
and the like (13) miso (fermented soybean) (14) baked soybean (15) canned orbottled soybean (16) frozen corn (17) canned or bottled corn (18) frozen potato
Other: Target products difficult to classify due to the variations in form.
For the following items, choose the protocol by consulting the above classification.
Products in which the major raw material is: a material listed in (1)-(18), soybean(for cooking), soybean powder, soybean protein, soybean in pods, soybean sprout,corn flour, corn grits (except corn flakes), corn (for cooking), potato (for cooking),
alfalfa, and sugar beet (for cooking).

３GM quicker 4 Manual Ver. 3.0

VI Protocol<Reagents, instruments etc., necessary in addition to the kit>
・Micropipette・Pipette tips
・50 ml Conical tube
・2.0 ml Microtube
・1.5 ml Microtube
・Food mill (Cutter mill etc.)
・Centrifuge
・Test tube mixer
・Incubator<Pretreatment method for food products>
In the food product inspection, to reduce sampling error, it is necessary to homogenize well sothat the food product is homogeneous. JAS analytical test handbook “Manual for testing /analyzing genetically modified food products” <3rd Edition> considers methods by which thesample is pulverized after adding sterilized water of the same weight or by which the sample isdried by freeze drying and then pulverized, but mainly describes the methods by which sterilized
water is added and the sample is homogenized with a cutter mill or the like.
In this kit, protocol 1 describes the method developed for food products that have been dried byfreeze-drying or the like, and protocol 2 describes the method developed for liquid food products
and homogenized food products prepared by adding sterilized water to a sample.

４GM quicker 4 Manual Ver. 3.0

<Protocol 1: DNA extraction method from dried foods>(1) Weigh 1.0 g of the pulverized sample in a 50 ml conical tube, and add 4.0 ml of GE1 Buffer, 10
μl of RNase A, 2 μl of α-Amylase and 20 μl of Proteinase K, respectively.

Conduct flash

centrifugation (momentary centrifugation) in order to have any sample and Buffer on the sides
collect at the bottom of the tube and then mix well with a Test tube mixer (30 seconds or longer).
Note 1(2) Heat at 65ºC for 30 min.

Stir at the highest speed for 10 sec at 10 minutes intervals with theTest tube mixer.
(3) Add 400 μl of GE2-M Buffer and stir well with the Test tube mixer.
(4) Centrifuge (≥ 4K x g, 10 min, 4ºC). Note 2(5) Transfer 800 μl of the supernatant to a fresh 2.0 ml microtube. Note 3(6) Add 600 μl of GB3 Buffer and mix well by inverting the tube 10-12 times.
(7) Centrifuge (≥ 10K x g, 5 min, 4ºC) and recover as much of the supernatant as possible into a 2.0ml microtube. Note 4(8) Transfer 700 μl of the supernatant of (7) to a Spin Column, centrifuge (≥ 10K x g, 1 min, 4ºC)
and discard the filtrate.
(9) Transfer the entire residual supernatant of (7) to the Spin Column of (8), centrifuge (≥ 10K x g, 1min, 4ºC) and discard the filtrate.
(10) Add 600 μl of GW Buffer to the Spin Column and then centrifuge (≥ 10K x g, 1 min, 4ºC)
and discard the filtrate.
(11) Transfer the Spin Column to a fresh 1.5 ml microtube.
(12) Add 50 μl of TE (pH8.0) dropwise to the center of the membrane and then leave standingfor 3 min at room temperature.
(13) Centrifuge (≥ 10K x g, 1 min, 4ºC) and recover the filtrate.

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GM quicker 4 Manual Ver. 3.0

<Protocol 2: DNA extraction method from foods containing large amount of water>(1) Weigh 1.0 g of the pulverized sample in a 50 ml conical tube, and add 1.0 ml of GE1 Buffer, 10μl of RNase A, 2 μl of α-Amylase and 20 μl of Proteinase K, respectively.

Conduct flash

centrifugation (momentary centrifugation) in order to have any sample and Buffer on the sides
collect at the bottom of the tube and then mix well with a Test tube mixer (30 seconds or longer).
Note 1(2) Heat at 65ºC for 30 min.

Stir at the highest speed for 10 sec at 10 minutes intervals with theTest tube mixer.
(3) Add 200 μl of GE2-M Buffer and stir well with the Test tube mixer.
(4) Centrifuge (≥ 4K x g, 10 min, 4ºC). Note 2(5) Transfer 800 μl of the supernatant to a fresh 2.0 ml microtube. Note 3(6) Add 600 μl of GB3 Buffer and mix well by inverting the tube 10-12 times.
(7) Centrifuge (≥ 10K x g, 5 min, 4ºC) and recover as much of the supernatant as possible into a 2.0ml microtube. Note 4(8) Transfer 700 μl of the supernatant of (7) to a Spin Column, centrifuge (≥ 10K x g, 1 min, 4ºC)
and discard the filtrate.
(9) Transfer the entire residual supernatant of (7) to the Spin Column of (8), centrifuge (≥ 10K x g, 1min, 4ºC) and discard the filtrate.
(10) Add 600 μl of GW Buffer to the Spin Column and then centrifuge (≥ 10K x g, 1 min, 4ºC)
and discard the filtrate.
(11) Transfer the Spin Column to a fresh 1.5 ml microtube.
(12) Add 50 μl of TE (pH8.0) dropwise to the center of the membrane and then leave standingfor 3 min at room temperature.
(13) Centrifuge (≥ 10K x g, 1 min, 4ºC) and recover the filtrate.

６GM quicker 4 Manual Ver. 3.0

(Note 1)

If the stirring is insufficient, the DNA yield is decreased markedly. Vertically apply
the tube to the Test tube mixer and make sure to stir for 30 sec. If the stirring is
insufficient, further stir for 30-60 sec.

(Note 2) Confirm the maximum revolving speed of the rotor of the refrigerated centrifuge and themaximum g-force resistance of the conical tube to be used.

(Note 3) Recover the supernatant in such a way that precipitates and film-like floating objects are
not taken if at all possible.
(Note 4) Recover the supernatant to a fresh 2.0 ml tube in such a way that precipitates andfilm-like floating objects are not taken if at all possible.

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GM quicker 4 Manual Ver. 3.0

VII Data Collection
1. Absorption spectra of DNA extracted with this kit.
The absorption peak in the vicinity of A260 showed that DNA extracted with this kit ishigh purity.
0.35
0.3

Abs.

0.25
0.2
0.15
0.1
0.05
0220

240260280300320

Wavelength [ nm ]Fig. 1

Absorption spectra of DNA extracted from soybean powder (Protocol 1)

0.25

Abs.

0.2
0.15
0.1
0.050220240260

280300320

Wavelength [ nm ]Fig. 2

Absorption spectra of DNA extracted from soy milk (Protocol 2)

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GM quicker 4 Manual Ver. 3.0

0.9
0.8
0.7
Abs.

0.6
0.5
0.4
0.3
0.2
0.1
0220240260

280300320

Wavelength [ nm ]Fig. 3

Absorption spectra of DNA extracted from corn soup powder (Protocol 1)

0.8
0.7
0.6
Abs.

0.5
0.4
0.3
0.2
0.10220240260

280300320

Wavelength [ nm ]Fig. 4

Absorption spectra of DNA extracted from potato snacks (Protocol 1)

９GM quicker 4 Manual Ver. 3.0

2. Agarose gel electrophoresis of DNA extracted with this kit.
DNA could be extracted from processed food products of soybean (soybean powder: Protocol 1
and soy milk: Protocol 2).

M

1

2

3

4

5

6

M

Lane M : OneSTEP Marker 6 (λ/ StyI digest)
Lane 1, 2, 3 : DNA derived from soybean powder
Lane 4, 5, 6 : DNA derived from soy milk
1/10 amount of DNA solution (5 μl) extracted with this kit was electrophoresed in 1% Agarose S.

DNA could be extracted from processed food products of corn (corn soup powder: Protocol 1 andcanned whole corn: Protocol 2).

M123456M

Lane M : OneSTEP Marker 6 (λ/ StyI digest)
Lane 1, 2, 3 : DNA derived from corn soup powder
Lane 4, 5, 6 : DNA derived canned whole corn
1/10 amount of DNA solution (5 μl) extracted with this kit was electrophoresed in 1% Agarose S.

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GM quicker 4 Manual Ver. 3.0

3. Detection of endogenous genes by PCR
PCR was performed using endogenous potato DNA UGPase oligonucleotide (Code No.:310-05991) and the DNA solution derived from potato snacks extracted with this kit as a template,
The PCR conditions were as follows: 95°C, 10 min → (95°C, 30 sec → 60°C, 30 sec → 72°C,30 sec) x 40 Cycles → 72°C, 7 min.

M

1234

M

111 bpLane M : OneSTEP Marker11 (pUC19/MspI digest)
Lane 1 : Negative control (TE solution)
Lane 2-4 : Potato snacks
A portion of the PCR product (5 μl) was electrophoresed in 3% Agarose 21 gel.
4. Detection of endogenous gene by real-time PCRQuantitative tests of DNA extracted with this kit from rice noodle and rice paper were performed
using rice endogenous DNA PLD oligonucleotide set 2 (Code No.: 312-07531) (n=3).

②Delta Rn①④③

Amplification curve by ABI7500 Fast System (Base Line 3-15, Th.Line=0.128)(1) Rice noodle DNA (190,000 copies/2.5 μl)(2) 10-fold diluted rice noodle DNA (12,800copies/2.5 l) (3) Rice paper DNA (1,840 copies/2.5 μl) (4) 10-fold diluted rice paper DNA(147 copies/2.5 μl)
The processed rice DNA extracted with this kit can be quantitated by real-time PCR.

１１GM quicker 4 Manual Ver. 3.0

VIII

Troubleshooting

TroubleAfter centrifugation,
sufficient amount of
the supernatantcannot be recovered.

Spin column isclogged.

Cause

Suggestion

Not enough amountof GE1 Buffer for thesample.

Reduce the amount of the sample or increase the amount of GE1Buffer.

Insufficient
centrifugal force
The temperature of
the crude extract is
not decreased from
65°C to room
temperature.
Precipitates andfloating objects on
the surface of thesupernatant are
mixed in.

It may be effective to decrease the temperature by cooling in ice
cold water for several minutes immediately before thecentrifugation to recover the supernatant of (5).
To avoid the precipitates or floating objects being mixed in,transfer the supernatant of (5) to a 1.5 ml or 2.0 ml microtube andcentrifuge again at a higher g, and then recover the supernatantagain and add GB3 Buffer.
Precipitates may float when left standing after centrifugation.
When the precipitates are floating, centrifuge again before
recovering the supernatant.

Insufficient sample
crushing

Crush the sample as fine as possible so as to be homogeneous.

Low extraction
efficiency

Mix well with the Test tube mixer after the addition of GE1 Bufferand enzyme. During the heating reaction, the viscosity isdecreased and the efficiency of stirring increases, so mix well.

Insufficient elution

When centrifugation-elution is performed immediately after theaddition of TE (pH8.0) to the Spin Column, the DNA yield may bevariable. Allow sufficient elution by standing at room
temperature for a few minutes, and then centrifuge.

Amount of DNA in
the sample is smallUse 2 or more samples and treat them separately until the recoveryof the supernatant of (7) at the step of centrifugation after theaddition of GB3 Buffer. Then, combine the recovered
supernatant and apply the whole mixture to a Spin Column in
multiple fractions. Thereby, increased DNA yield is expected.

The temperature of
the enzyme treatment
is too low or toohigh.

Adjust the temperature as described in the manual(65°C). Also, in the heating process, it is recommended
to use a water bath or a heat block having a good heat
conduction efficiency.

Loss of degrading
enzyme activity

Store Proteinase K and α-Amylase at -20°C.

RNase A isinactivated

GE1 buffer and RNase A cannot be stored as a mixture.
them separately.

Low yield ofDNA

High RNA
contamination

Increase the centrifugal g or the centrifugal time.

StorePerform PCR after diluting with sterilized water or the like.
DNA cannot beamplified by PCR

PCR inhibitor mayexist.

When the coloring to the spin column is due to the dye derived
from the sample, it may be effective i) to perform process of (10)of washing with GW Buffer twice, or ii) to wash with 80% ethanol
after the washing process of (10).

１２GM quicker 4 Manual Ver. 3.0

IX Related Products
Code No.

製品名

包装単位

317-06361
310-06591
311-07241
319-07161
317-07341
314-06371
311-06381
318-06391
311-06641
318-06651
315-06661
312-06671

GM quicker
GM quicker 2
GM quicker 3
GM quicker 96
On-Site Column Set for GM quicker
GE1 Buffer
GE2 Buffer
RNase A (100 mg/ml)
GW Buffer
GE2-K Buffer
GB3 Buffer
GM quicker 2 Enzyme Set(Proteinase K 2 ml, α-Amylase 0.2 ml)
Collection Tube

50 reactions50 reactions
50 reactions
96well-plate×420 reactions
500 ml200 ml
0.5 ml×540 ml×2
100 ml12.5 ml×2
1 Set

319-08141

１３50 tubes×2

GM quicker 4 Manual Ver. 3.0

X Reference1) Yasutaka Minegishi, Junichi Mano, Yasuo Kato, Kazumi Kitta, Hiroshi Akiyama
and Reiko Teshima (2013) Japanese Journal of Food Chemistry and Safety, in press

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GM quicker 4 Manual Ver. 3.0

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

http://www.nippongene.com/