DNA extraction kit from plants, yeasts and bacteria

ISOPLANT
Manual (8th edition)

Code No. 314-02731 For 100 extractions
Code No. 310-02733 For 20 extractions

NIPPON GENE CO., LTD.

Table of contents

I

Product description

1

IIContents of kit

1

III

Storage

1

IV

Precautions

2

VProtocol

3

VI

Troubleshooting

5

VII Q & A

5

VIII Data collection

6IX

1. DNA extracted from plants

6

2. DNA extracted from bacteria

7

3. DNA extracted from yeasts

8

4. DNA extracted from actinomycete and fungi

8References

8

ISOPLANT Manual (ver.8)
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IProduct description

ISOPLANT is a kit for extracting DNA from plants, yeasts and bacteria in a short period of time.
ISOPLANT 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. Since the sample canbe solubilized in the presence of a surface-active agent, DNA can be extracted without grinding,especially in the case of plants. Therefore, this is very convenient when processing many samples.
DNA can be extracted without pretreatment not only from dicot plants, but also monocot plants.
The obtained DNA can be used for PCR and restriction enzyme reactions as-is.

IIContents of kit(For 100 extractions)
Solution I*： Extraction Buffer(For 20 extractions)

30 ml

6 ml

Solution II ： Lysis Buffer

15 ml

3 ml

Solution III ： Sodium Acetate (pH 5.2)

15 ml

3 ml

TE： 10 mM Tris-HCl (pH 8.0), 1 mM EDTA

10 ml

2 ml

RNase A： 1 mg/ml

100 μl20 μl* White precipitates may appear in the Solution I but this does not affect the quality.

In such cases,use the solution after completely dissolving the crystals in a water bath at about 37°C.

Also, handle

the solution with care because the Solution I contains a protein denaturant and the like.

If theSolution I enters eye or contacts skin, immediately wash well with large amounts of water.

IIIStorage

Chilled storage (4°C)
All the reagents except 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).

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ISOPLANT Manual (ver.8)
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IVPrecautions○ 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 hazardousmaterial, 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.
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 inthe manual.
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VProtocol

Sample *1)
Solution I

300 μl *2)

Vortex

1-2 sec

Solution II

150 μl *3)

Vortex *4)

5-6 sec

50°C15 min *5)

Solution III

150 μl *6)

Vortex

1-2 ses

On ice15 min *7)

12 K×g, 4°C, 15 min
Aqueous phase *8)
2 - 2.5 volume of ethanol *9)
Mix
12 K×g, 4°C, 10 min
Precipitates *10)
70% ethanol wash
Air dry *11)
TE (pH 8.0)

50 μl

DNA solution *12)
*1)

*2)
*3)

In case of plants, cut 0.01-0.1 g of the sample into 3-5 mm squares with scissors andthen use. Although the volume may change depending on the variety of plant, it ispreferable to immerse the sample entirely in the lower layer (Solution II) when Solution IIis added.
Also, use fresh samples as much as possible. Even stored frozen, the recovery rate ofDNA may be lower when the storage time is long.
In the case of yeast, bacteria and the like, use the precipitates (～0.03 g) obtained bycentrifuging a 1.5 ml culture.
Since Solution I contains SDS, white crystals may form when stored at a lowtemperature. In such a case, use Solution I after completely dissolving in a water bathat about 37°C.
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 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.
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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） White turbidity is formed by vortexing.
*5） 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 (Solution I containing surface-active agent). For plant samples, themorphological changes are normally not visible.
*6） By adding Solution III, white turbidity is formed.
*7） White turbidity is further formed when cooled on ice.
*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. In order to prevent
physical cleavage of DNA., cut the pipette tips.
*9） In situations where PCR and restriction enzyme reactions do not proceed well,reactions may be improve by performing ethanol precipitation twice. When performing
the ethanol precipitation, use ethanol cooled at -20°C, and centrifuge immediately after
the addition. If the mixture is left standing at -20°C for a long period of time, this may
cause co-precipitation of contaminants.
*10）DNA precipitates may be invisible. Depending on the variety of the sample,precipitates other than DNA may be formed. For example, rice plant may cause brown
precipitates, and spinach may cause white precipitates which are different from DNA.
*11）Since completely dried precipitates become very difficult to dissolve, make sure not todry too much.
*12）Add TE when non-DNA debris precipitates out (see *10) ). If it does not dissolve
completely by adding TE, let it stand on ice for a while, and collect the supernatant after
brief centrifuge. Note that if precipitates are found to have brownish color, see VItroubleshooting in p.5. When RNA in the DNA solution is to be removed, add attached
RNase A to a final concentration of 10-20 μg/ml and react at 37°C for 30 min. If
necessary, perform phenol/chloroform treatment after the reaction.

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VI Troubleshooting
TroubleCountermeasure

White crystals appear in theSolution I.
DNA is brown after ethanol
precipitation and centrifuging.
Does it have any influence on
PCR?

Dissolve crystals completely in a water bath at about 37°C, stir
so that the content becomes homogeneous and then use it.
When a brownish color is found in precipitates, contamination
by polyphenol, which is rich in plants, may be considered.
This substance inhibits PCR, but the degree of inhibition may
be lowered by adding 2-Mercapto-ethanol at 2% (finalconcentration) to Solution I beforehand.
Dissolve gelatinous DNA into TE or H2O.
Add the High-Salt Precipitation Solution* at a half volume of thisdissolved solution and stir well. Perform isopropanol precipitation
by adding isopropanol at an equal volume of the High-Salt
Precipitation Solution and stir well. Isopropanol to be added does
not need to be cooled. Immediately after the addition, centrifuge
at 10 K×g for 15 min to obtain DNA precipitates.
* High-Salt Precipitation Solution: Code No. 313-06341(1.2 M NaCl, 0.8 M Sodium citrate）

When samples such as legume
plants containing a large amount ofsugar
areused,gelatinous(transparent) DNA precipitates areobtained.

Yield is low.

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

・ Samples should be as fresh as possible.
・ mince the sample into 3 mm×3 mm or smaller pieces as
finely as possible.
・ Scale up.
・ Mix by inverting a few times during the 50°C incubation
period.
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.
An improvement may be made by repeating the ethanol
precipitation twice. In this case, use ethanol cooled at -20°Cand centrifuge immediately.

VII Q & A
Q1. In the case of plant samples, the morphology of the sample does not change after adding Solution II.
A1. When processed in accordance with the normal protocol, if this is left immersed in Solution II for a long periodof time, the morphology changes even though the change may not be determined by eye. However, whenprocessed to this condition, the DNA solution finally obtained may contain a large amount of contaminants.
Q2. In the case of stiff leaves such as fruit trees, the DNA yield is low using the normal protocol.
A2. DNA may be extracted by freezing the sample with liquid nitrogen and pulverizing beforehand.
Q3. Can DNA be isolated from animal tissues/cells?
A3. No, generally speaking this kit is not suitable for that, although it has been confirmed that DNA can be isolated
from shrimps. At the moment, DNA extraction using this kit is confirmed for plants, yeasts, bacteria, fungi and thelike. (See p. 6, VIII Data collection.)-5-

ISOPLANT Manual (ver.8)
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VIII Data collection【 DNA extracted from plants 】《 Yield and purity of extracted DNA 》
ISOPLANT
yield (μg/g)
A 260/280
100-1201.880-120
1.8
4-20
1.8
10-20
1.8
10-20
1.8

Arabidopsis thaliana
spinachtobaccotulip
rice

CTAB* methodyield (μg/g)
A 260/280
15-30
1.830-50
1.8
60-80
1.7
10-20
1.8
10-20
1.7

* CTAB: cetyltrimethylammonium bromide《 Restriction enzyme reaction of extracted DNA 》
It has been confirmed that the extracted DNA can be cut with restriction enzymes (EcoR I and Hind III) using
2-20 times of the quantity.
1Mab2cab3cab4cabc

Mlane
1 spinach
2 Arabidopsis thaliana3 tobacco
4 ricea: intactb: EcoR I digest
c: Hind III digest
M: Marker 6 (λ/Sty I digest)《 PCR of extracted DNA 》
It has been confirmed that the specific band can be amplified by PCR using the extracted DNA as a template
and using the primers for amplifying the tobacco ribulose-1,5-diphosphate carboxylase large subunit gene.
M

12

3

45lane1 spinach
2 Arabidopsis thaliana
3 tobacco
4 tulip
5 rice
M: Marker 6 (λ/Sty I digest)-6-

ISOPLANT Manual (ver.8)
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【 DNA extracted from bacteria 】《 Yield and purity of extracted DNA 》
ISOPLANT
yield (μg/g)
A 260/280
125 – 500
1.8250 – 500
1.81001.8
125 – 500
1.8250 – 1000
1.880 – 125
1.81251.95001.81001.8

Anabaena variabilis
Bacillus amyloiquefaciens
Bacillus subtilis
Escherichia coli
Haemophilus influenzae
Pseudomonas aeruginosa
Staphylococcus aureus
Thermus aquaticus
Xanthomonas holcicola

phenol/chloroform method
yield (μg/g)
A 260/280
60 – 500
1.71000
1.8－－
250
1.8
250
1.7－－
125
1.75001.75001.7《 Restriction enzyme reaction of extracted DNA 》
It has been confirmed that the extracted DNA can be cut with restriction enzymes (EcoR I and Hind III) using
2-20 times of the quantity.
Ma1bc

2baca3bclane
1 Escherichia coli JM109
2 Staphylococcus aureus 3A
3 Pseudomonas aeruginosa
a: intactb: EcoR I digest
c: Hind III digest
M: Marker 6 (λ/Sty I digest)《 PCR of extracted DNA 》
It has been confirmed that the specific band can be amplified by PCR using the extracted DNA as a template.
M

1234

lane
1 Escherichia coli JM109
2 Bacillus amyloiquefaciens
3 Bacillus subtilis
4 Staphylococcus aureus 3A
M: Marker 6 (λ/Sty I digest)-7-

ISOPLANT Manual (ver.8)
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【 DNA extracted from yeasts 】《 Yield and purity of extracted DNA 》

Saccharomyces cerevisiae
Shizosaccharomyces pombe
Phichia pastoris

yield (μg/mg)
0.504
0.348
2.523

M

12A 260/280
1.83
1.79
1.87

3lane
1 Saccharomyces cerevisiae
2 Shizosaccharomyces pombe
3 Phichia pastoris
M: Marker 6 (λ/Sty I digest)【 DNA extracted from actinomycete and fungi 】《 Yield and purity of extracted DNA 》
yield (μg/g)
10.8
21.2
3.3
0.8
8.0
34.8
23.0
32.8
36.1
20.5
36.0
6.8

Mycrobacterium phlei
Nocardia asteroides
Streptomyces albus
Streptomyces sp.2-1
Streptomyces sp.MY-31
Neurospora sitophila
Rhizopus nigricans
Penicillium chrysogenum Q176
Aspergillus awamori
Aspergillus japonicus
Aspergillus niger
Aspergillus oryzae

A 260/280
1.79
1.92
2.22
2.00
2.09
1.73
2.04
1.80
1.62
1.90
1.73
2.04

IX References1) Anil K.Jhingan,Methods in Molecular and Cellular Biology,3,15(1992)
2) Heng,Z.et al,Nucleic Acids Research,21(22),5279(1993)-8-

ISOPLANT Manual (ver.8)
120521KH

NIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA 930-0834
JAPANTel
076-451-6548
URL
https://www.nippongene.com/

ISOPLANT Manual (ver.8)
120521KH