DNA extraction kit from plants, yeasts and bacteria

ISOPLANT II
Manual (Third edition)

Code No. 310-04151 For 100 extractions
Code No. 316-04153 For 20 extractions

NIPPON GENE CO., LTD.

Table of contents

I

Product description

1

II

Contents of kit

1

III

Storage

2

IV

Precautions

2

V

Protocol3VI

1. Plant

3

2. Yeasts and bacteria

6

Troubleshooting

8

VII Data collection

9

1. RAPD analysis using DNA extracted from plant leaves

9

2. Restriction enzyme reaction of DNA extracted from plant leaves

9

3. Restriction enzyme reaction of DNA extracted from bacteria and yeasts

10

4. Comparison of purity

10

between ISOPLANT II and a conventional product (ISOPLANT)VIII References

11

ISOPLANT II Manual (ver.3)
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I

Product description

ISOPLANT II is a kit for extracting DNA from plants, yeasts and bacteria in a short period of time.
Plant tissues contain more of polysaccharides and polyphenols compared to animal tissues, andthese may contaminate extracted DNA. These substances inhibit restriction enzyme reactions and
PCR making the quantitative determination of DNA difficult.
ISOPLANT II disrupts cell walls, cell membranes, nuclear membranes and the like with benzyl
chloride, which is the main component of the Solution II of the ISOPLANT II as well as theconventional ISOPLANT, and solubilizes in the presence of a surface-active agent. Further, in the
ISOPLANT II, the reagents and protocol have been improved so that the inhibitors such aspolysaccharides and polyphenols are effectively removed. Also, DNA can be extracted not only
from herbaceous plants but also from woody plants from which DNA extraction has been difficult
until now, and the obtained DNA can be used for PCR and restriction enzyme reactions.

II

Contents of kit(For 100 extractions)(For 20 extractions)

Wash Buffer：

100 ml20 ml

Solution I *1

：

30 ml

6 ml

*2

：

15 ml

3 ml

Solution III-A *3

：

10 ml

2 ml

Solution III-B

：

12 ml

2.5 mlTE (pH8.0)

：

10 ml

2 ml

RNase A (1 mg/ml) ：100 μl

20 μl

Solution II

Manual*1

White precipitates may appear in the Solution I but this does not affect the quality.

In suchcases, use the solution after completely dissolving the crystals in a water bath at about 50°C.
Also, handle the solution with care because the Solution I contains a protein denaturant and thelike.
water.
*2

If the Solution I enters eye or contacts skin, immediately wash well with large amounts ofFurther, if there are other abnormal symptoms, immediately consult a physician.

Benzyl chloride is the main component of the Solution II.

Benzyl chloride is a designatedhazardous material under the Fire Service Act (group 4 hazardous material, group 2 petroleum,
hazard degree III, flammable, keep away from flames). When handling, make sure to read theprecautions in p. 2, Section IV.
*3

The Solution III-A is a white suspension. When the Solution III-A is evaporated to dryness andonly the white solid phase remains, dissolve the solid phase using sterilized distilled water (fordetails, see the troubleshooting instructions in p. 8, Section VI).

Note) When DNA is extracted from plant samples, 2-Mercaptoethanol (designated hazardous material
under the Fire Service Act) and in some cases NaBH4 (designated hazardous material under theFire Service Act) are required separately.

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III

Storage

Chilled storage (4°C)
All the reagents except Wash Buffer and RNase A can be stored at room temperature.
Further, when the kit is not to be used for a long period of time, store RNase A frozen (-20°C).

IV

Precautions○ This product is a reagent for research and cannot be used for other purposes such as forpharmaceutical purposes. Also, this product should be handled only by persons who
have basic knowledge of reagents.
○ The main component of the Solution II included in this product is benzyl chloride. Benzyl
chloride is a designated hazardous material under the Fire Service Act (group 4 hazardous
material, group 2 petroleum, hazard degree III, flammable, keep away from flames).
Handle with care. See the following for hazardous effects and handling precautions.
· Hazardous effects···
This product has extremely strong irritant properties and tearing properties to skinand mucous membranes, and may cause chemical burn on contact. Inhaling
vapor at high concentrations may cause symptoms such as coughing, headaches,burning sensations, hypoesthesia, consciousness loss, tremor of limbs andparalysis.
· Handling precautions···
Keep away from flames and avoid contact with strong oxidants.
Install a local ventilating apparatus in the area where the reagent is handled, andclose the lid of the container tightly after use.
Wear proper protective gears so as not to inhale or allow contact to eye, skin andclothing.
If the eye, skin or the like makes contact with the reagent, perform the following
procedures.
If the reagent enters eye :Immediately wash with a large amount of water for not less than 15 min.
Skin contact :Wash with a large amount of water using soap.
Inhalation :Rest and stay warm in a well-ventilated area.
Accidental ingestion :Rinse out the mouth, drink activated charcoal suspended in water and consult aphysician.

Since the mucous membrane in the stomach and the like isdamaged, do not force vomiting.

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Also, if there are other abnormal symptoms, immediately consult a physician.
For storage, avoid direct sunlight, keep in a cool dark place and avoid contact with air byhermetically sealing the product. Further, if a minute amount of a metal such as iron is mixed
in the container, a strong degradation reaction may occur, causing break-up of the container.
○ Handle this product in accordance with the descriptions in the manual. We cannot takeresponsibility for problems caused if the product is not handled according to the descriptions in
the manual.

V

Protocol1. Plant■ Reagent preparation: prepare the following reagents (A, B, C) when extracting DNA from a plant
sample.

Prepare reagents (A, B, C) immediately before use in the amount required for extraction.
Use up all of the reagents because storage of the reagent may cause decrease in yield andcontamination of the enzyme reaction inhibitors.
A. Wash Buffer + 2-Mercaptoethanol (2-ME)
Add 2-Mercaptoethanol (required separately) to attached Wash Buffer to a final
concentration of 0.5 %.
B. Solution I + 2-Mercaptoethanol (2-ME)
Add 2-Mercaptoethanol (required separately) to attached Solution I to a final
concentration of 1 %.
(Note) 2-Mercaptoethanol is a designated hazardous material under the Fire Service Act. When handling, checkthe precautions.

Example: Plants with high polyphenol content
C. 1% NaBH4
Dissolve NaBH4 (Sodium tetrahydroborate: required separately) with sterilized water to
1 % (w/v).
(Note) Sodium tetrahydroborate is a designated hazardous material under the Fire Service Act. When handling,check the precautions.

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ISOPLANT II Manual (ver.3)
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Sample *1)

0.01-0.1 g
Wash Buffer + 2-ME

1 ml

Mix
14 K x g, 4°C, 10 min
Precipitates*2)

Solution I + 2-ME
Vortex300 μl *3)

1-2 sec
30 μl *4)

1% NaBH4
Mix

Treatment required for theplants containing muchpolyphenols.

150 μl *5)

Solution II
Vortex

10 sec50°C

10 min *6)

Solution III-A

100 μl *7)

Solution III-B

120 μlVortex

1-2 sec

On ice10 min14 K x g, 4°C, 10 min
Aqueous phase *8)
2 volume of ethanol (Store at room temperature)Mix
6 K x g, room temperature, 5 min *9)
Precipitates
70 % ethanolMix
6 K x g, room temp, 1 min
Precipitates
Air dry *10)
TE (pH 8.0)
DNA solution

*11)

*1)

Mince 0.01 g-0.1 g of the plants to 1 mm cubes with a cutter, or freeze/pulverize withliquid nitrogen.
The yield is higher when freezing/pulverization is performed.
Especially for woody plants, the yield can be very low without freezing/pulverization.
Immerse the plant sample in the Wash Buffer immediately after freezing/pulverization.
If left in the air for a long period of time, a browning reaction takes place due to oxidationof polyphenols causing problems for DNA extraction.

*2)

Remove as much of the supernatant as possible. If the minced sample is used, thesample floats even if centrifugation is performed and removing the supernatant may bedifficult. Dip the end of a pipette tip in the solution and suck out so that the remaining
amount of the solution is as small as possible.

*3） White crystals may appear in the Solution I. In such cases, dissolve the crystals
completely in a water bath at about 50°C, stir so that the content becomeshomogeneous and then use it. Also, since the Solution I contains protein denaturant
and the like, handle with care. If the Solution I enters eye or contacts skin, immediately
wash well with a large amount of water.

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*4） NaBH4 (sodium tetrahydroborate: designated as a hazardous material under the Fire
Service Act) is a strong reducing agent and used for suppressing oxidation ofpolyphenols contained in the plant tissues. When NaBH4 is added, bubbles areformed. Take care not to allow the bubbles to leak from the tube and do not let thecontents expand out from the tube when opening the cap.
These dangers can be avoided by quenching bubbles by lightly spinning down after
adding NaBH4 and before and after the 50°C incubation.
*5） The main component of the Solution II included in this product is benzyl chloride.
Benzyl chloride is a designated hazardous material under the Fire Service Act.
Handle with care. (See p. 2, IV Handling precautions.)
If eye, skin or the like make contact with the reagent, perform the following procedures
immediately.
If the reagent enters eye :Immediately wash with a large amount of water for not less than 15 min.
Skin contact :Wash with a large amount of water using soap.
Inhalation :Rest and stay warm in a well-ventilated area.
Accidental ingestion :Rinse out the mouth, drink activated charcoal suspended in water and consult aphysician. Since the mucous membrane in the stomach and the like is damaged, do
not force vomiting.
Also, if there are other abnormal symptoms, immediately consult a physician.
*6） Cell walls, cell membranes, nuclear membranes and the like are disrupted by benzyl
chloride, which is the main component of the Solution II, and DNA is dissolved intoaqueous phase. For plant samples, the morphological changes are normally not
visible.
*7） Shake the Solution III-A well to homogeneity and suck out with a pipette tip with a cutedge.
*8） Avoid taking substances other than the aqueous phase, such as benzyl chloride in theorganic phase, the solid white substance found after the centrifugation and floating
pieces of the plant, as much as possible. If substances other than the aqueous phaseare mixed in, centrifuge again and collect only the aqueous phase. When the aqueous
phase is cloudy white, add Solution III-A and B again, mix, centrifuge (14 K x g, 4°C, 10min) and use the aqueous phase. (See p. 8, VI Troubleshooting.)*9） Centrifuge immediately after adding ethanol stored at room temperature.
standing at a low temperature such as -20°C, contaminants may be mixed in.

If left*10）Since completely dried precipitates become very difficult to dissolve, make sure not to
dry too much.
*11） When insoluble substances exist in the DNA solution, dissolve DNA by standing on ice
for a while, centrifuge lightly and use the supernatant. When RNA in the DNA solution
is to be removed, add attached RNase A to a final concentration of 10-20 μg/ml andreact at 37°C for 30 min. If necessary, perform phenol/chloroform treatment after thereaction. (See p. 8, VI Troubleshooting.)-5-

ISOPLANT II Manual (ver.3)
111028KH2. Yeasts and bacteria■ Protocol

Sample (1.5 ml culture)
6 K x g, room temperature, 5 min
Precipitates
300 μl *1)

Solution I
Vortex *2)

150 μl *3)

Solution II
Vortex

10 sec50°C

10 min *4)

Solution III-A

100 μl *5)

Solution III-B

120 μlVortex

1-2 sec

On ice10 min14 K x g, 4°C, 10 min
Aqueous phase *6)
2 volumes of ethanol (Stored at room temperature)Mix
6 K x g, room temperature, 5 min
Precipitates*7)

70% ethanol

1 ml

Mix
6 K x g, room temperature, 1 min
Precipitates
Air dry *8)
TE (pH 8.0)
DNA solution

*9)

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*1） White crystals may appear in the Solution I. In such cases, dissolve the crystals
completely in a water bath at about 50°C, stir so that the content becomeshomogeneous and then use it. Also, since the Solution I contains protein
denaturant and the like, handle with care. If the Solution I enters eye or contacts
skin, immediately wash well with a large amount of water.
*2） Suspend the bacteria body well.
*3） The main component of the Solution II included in this product is benzyl chloride.
Benzyl chloride is a designated hazardous material under the Fire Service Act.
Handle with care. (See p. 2, IV Handling precautions.)
If eye, skin or the like make contact with the reagent, perform the following
procedures immediately.
If the reagent enters eye :Immediately wash with a large amount of water for not less than 15 min.
Skin contact :Wash with a large amount of water using soap.
Inhalation :Rest and stay warm in a well-ventilated area.
Accidental ingestion :Rinse out the mouth, drink activated charcoal suspended in water and consult aphysician. Since the mucous membrane in the stomach and the like is damaged, do
not force vomiting.
Also, if there are other abnormal symptoms, immediately consult a physician.
*4） Cell walls, cell membranes, nuclear membranes and the like are disrupted by benzyl
chloride, which is the main component of the Solution II, and DNA is dissolved intoaqueous phase.
*5） Shake well the Solution III-A to homogeneity and suck out with a pipette tip with a cutedge.
*6） Avoid taking substances other than the aqueous phase, such as benzyl chloride in
the organic phase and the solid white substance found after the centrifugation, as
much as possible. If substances other than the aqueous phase are mixed in,centrifuge again and collect only the aqueous phase.
*7） DNA precipitates may not be visible.
*8） Since completely dried precipitates become very difficult to dissolve, make sure not
to dry too much.
*9） When insoluble substances exist in the DNA solution, dissolve DNA by standing on
ice for a while, centrifuge lightly and use the supernatant. When RNA in the DNA
solution is to be removed, add attached RNase A to a final concentration of 10-20μg/ml and react at 37°C for 30 min. If necessary, perform phenol/chloroform
treatment after the reaction. (See p. 8, VI Troubleshooting.)-7-

ISOPLANT II Manual (ver.3)
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VI Troubleshooting

TroubleCountermeasure

White crystals appear in the
Solution I.
The Solution III-A is evaporated todryness leaving only white solid
phase.

Dissolve crystals completely in a water bath at about 50°C, stir
so that the content becomes homogeneous and then use it.
Dissolve using sterilized distilled water.
(1) Add H2O until the entire solid phase is immersed.
(2) Since the solid phase has water absorbing property, wait
for a while.
(3) When the surface is dried, add H2O again.
(4) Repeat H2O addition and water absorption until entire
surface of the solid phase is covered by H2O.
Depending on the plant species, some sample may not be
precipitated by the centrifugation. In such cases, dip the endof a pipette tip into the solution and suck out. Sometimes
pieces of the plant may be sucked out together but this isunavoidable.
After transferring the supernatant to another tube, add 100 μl of
the Solution III-A and 120 μl of the Solution III-B again, mix andcentrifuge (14 K x g, 4°C, 10 min), and use the supernatant.
(▼)
If the precipitates are found to have brown coloring, perform
re-extraction. In such cases, try the following treatments inthe extraction procedure.
(1) Perform NaBH4 treatment. (See p. 3.)(2) If 2-Mercaptoethanol was not added to the Wash Bufferand Solution I, then add 2-Mercaptoethanol. (See p. 3.)(3) Incubate at 50°C for 10 min after adding the Wash Buffer +2-ME to the plant sample.
(4) Perform (▼) described above.
・ When the precipitates are insoluble in TE, centrifuge anduse the supernatant. (▲)
・ Try (▼) described above in the extraction procedure.
Leave standing at 4°C overnight or at 50°C for 1 hr.
Pipetting may be performed but the obtained DNA may be
broken.
・ Samples should be as fresh as possible.
・ Use frozen pulverized sample.
If freezing andpulverization cannot be performed, mince the sample asfinely as possible.
・ Scale up.
・ Make the Solution II treating time longer. Keep in mind
that this may also increase contamination by foreign
matter.
Add the attached RNase A to the obtained DNA solution to a
final concentration of 10-20 μg/ml and react at 37°C for 30 min.
If necessary, perform phenol/chloroform treatment after thereaction.
・ When the DNA solution is colored, try (▼) describedabove. If white precipitates, which are different from
DNA, are obtained, try (▲) described above.
・ Inhibition of PCR can be reduced by decreasing theamount of the template. (1/10 dilution etc.)
Use a pipette tip with a cut edge to prevent physical breakdownof DNA during the extraction process.

It is difficult to collect thesupernatantbecause the plant
sample is floating after thecentrifugation after adding the
Wash Buffer + 2-ME.
The supernatant after adding the
Solution III-A, B and centrifuging isturbid or colored.
DNA is brown after ethanol
precipitation and centrifuging.

White precipitates other than DNA,are formed.
DNA precipitate is not dissolved.

Yield is low.

A large amount of RNA iscontaminating the obtained DNA
solution.
No amplification is found when
PCR is performed using theobtained DNA as a template.

Obtained DNA is broken down.

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VII Data collection[ RAPD analysis using DNA extracted from plant leaves ]RAPD analyses were performed using 1/100 amount of DNA extracted from 0.1 g of respective
frozen pulverized leaves.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

lane 1. Water melon
lane 2. Cucumber
lane 3. Pumpkin
lane 4. Cornlane 5. Ricelane 6. Green onion
lane 7. Beanlane 8. Tobaccolane 9. Spinachlane 10. Ginkgo
lane 11. Applelane 12. Persimmon
lane 13. Peachlane 14. Japanese citronlane 15. Prune
lane 16. Pear

M : Marker 4 (φX174/Hae III)
Arbitrary Primer: AP-A-2 (TGGATTGGTC)Taq DNA polymerase: Gene Taq
3% Agarose 21[ RAPD-PCR ]・PCR Mixture
Template DNA
10×Gene Taq Universal Buffer
dNTP Mixture (2.5 mM each)
AP-A-2 primer (10 μM)
Gene Taq (5 units/μl)
H2O

・PCR Condition
1 μl
5 μl
4 μl
1 μl
0.25 μl38.75 μl
50 μl

95°C
40°C
72°C

3 min.
5 min.
1 min.

1 cycle15 sec.
2 min.
1 min.

25 cycles

↓
95°C
40°C
72°C
↓
72°C

5 min.

[ Restriction enzyme reaction of DNA extracted from plant leaves ]Restriction enzyme reactions (5-20 units) are performed using 1/20 of the amount of DNA extracted
from 0.1 g of respective frozen pulverized leaves.

1
2
3
4
5
6
7
8
M a b a b a b a b a b a b a b a blane 1. Tobaccolane 2. Spinachlane 3. Carrot
lane 4. Egg plantlane 5. Ricelane 6. Tealane 7. Japanese citronlane 8. AppleM : Marker 2 (λ/Hind III・EcoR I)
a : intact DNA
b : Hind III digest + RNase A
0.8% Agarose S(Note) The above data were obtained using DNA treated with NaBH4 when extracted.

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[ Restriction enzyme reaction of DNA extracted from bacteria and yeasts ]Restriction enzyme reactions (5-20 units) are performed using 1/20 amount of DNA extracted from
1.5 ml of respective liquid cultures.
1
2
3
4
5
6
7
M a b a b a b a b a b a b a blane 1.
lane 2.
lane 3.
lane 4.
lane 5.
lane 6.
lane 7.

Serratia marcescens
Moraxella bovis
Haemophilus influenzae cProteus vulgaris
Shizosaccharomyces pombe
Pichia pastoris
Saccaromyces cerevisiae

M : Marker 2 (λ/Hind III・EcoR I)
a : intact DNA
b : Hind III digest + RNase A
0.8% Agarose S[ Comparison of purity between ISOPLANT II and a conventional product (ISOPLANT) ]DNA was extracted from the same amount of samples of spinach and apple leaves using
ISOPLANT II and the conventional product (ISOPLANT), and the absorbance (200-300 nm) was
measured.
DNA extracted with ISOPLANT II demonstrates a smooth mound shape having a peak at 260 nm,
indicating that these are highly purified DNA preparations from which contaminants such aspolysaccharides and polyphenols have been removed.

■ Spinach■ Apple- 10 -ISOPLANT II Manual (ver.3)
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VIII References
1. Xing S. and Aharon G.: Analytical Biochemistry, 174, 650-657 (1988)
2. C. S. Kim, C. H. Lee, J. S. Shin, Y. S. Chung and N. I. Hyung:
Nucleic Acids Res., 25 (5) 1085-1086 (1997)- 11 -ISOPLANT II Manual (ver.3)
111028KHNIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA
930-0834 JAPANTel+81-76-451-6548
URL
https://www.nippongene.com/

ISOPLANT II Manual (ver.3)
111028KH