DNA Extraction Reagent

ISOGENOME
Manual Ver. 2

Code No. 314-08113 (100 ml)

NIPPON GENE CO., LTD.

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Product Description

ISOGENOME is reagent for the isolation of genomic DNA from tissues or cells. The
ISOGENOME procedure is based on the use of a guanidine-detergent lysing solution whichpermits selective precipitation of DNA from a cell lysate. The procedure can be completed in30 min with DNA recovery of 70-100%. The ISOGENOME protocol is fast and permits
isolation of genomic DNA from a large number of samples of small or large volumes. Also,ISOGENOME can be isolated of DNA and RNA from same sample by the use of combination
with RNA extraction regent ISOGEN II (Code No. 311-07361).
The isolated DNA can be used without additional purification for applications such as
Southern analysis, molecular cloning, and polymerase chain reaction (PCR).

IIComponents

Code No. 314-08113
・ISOGENOME

III

100 mlStorage

Store at room temperature.

IV-Precautions
This product is a reagent for research and cannot be used for other objectives. Also, thisproduct should be handled only by persons having a basic knowledge about reagents.

-ISOGENOME is include irritant, and should be handled with great care. When using, wear
appropriate protective gear (gloves, goggles, etc.).

-Handle this product in accordance with the descriptions in the manual.

-We are not responsible for problems caused if this product is not handled in accordancewith the manual.

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V Protocol
Materials Required but not Provided-

99.5-100% Ethanol-75% Ethanol-

8 mM NaOH (use within 1 month of preparation. Alternatively, dilute a stock solution of2-4 M NaOH solution that has been prepared within the past 6 months to a final
concentration of 8 mM NaOH)-1M HEPES solution (for neutralization)

Unless stated otherwise, the procedure is carried out at room temperature.

＜Protocol A: Extraction of Genomic DNA＞
Step 1．Lysis of Sample (Homogenization)
1. Add 1 ml of ISOGENOME to Sample (25-50 mg tissue, or up to 1×107 cells, either inpellet or in suspension (volume < 0.1 ml)).
＜Quantity of cell or tissue that can be treated with 1 ml of ISOGENOME＞
SampleSample Volume

liver, kidney, lungs, heart,tissue25-50 mgskeletal muscle
spleen, brain (soft tissues)5-10 mg

adherent cell

10 cm

cell pellet

1×107 cells (< 0.1 ml)

suspended cell

0.1 mlcell2Homogenization2-a)2-b)2-c)

2. Homogenize by gently and thoroughly.
a) For animal tissue, homogenize tissue samples in a hand-held glass/Teflon®
homogenizer. Use a loosely fitting homogenizer, with a tolerance of 0.1-0.15 mm orhigher. Homogenize 25-50 mg tissue in 1 ml of ISOGENOME by applying as few
strokes as possible. Typically, 5-10 strokes are required for complete
homogenization.
For small amounts (5-10 mg) of soft tissues, such as spleen or brain, can bedispersed into smaller fragments and lysed by repetitive pipetting with amicropipette.
b) For adherent cells, after removing the medium from the dish, add 1 ml ofISOGENOME per 10 cm2 (equivalent to 3.5 cm dish) directly and lyse by pipetting
several times with a micropipette.
3c) For suspended cells (cell pellet or cell suspension), after centrifugation to remove
the medium, add 1 ml of ISOGENOME to 1 x 107 cells (volume <0.1 ml) and lyse
cells by repeated pipetting.
3.

In the case of animal tissues, store the homogenate for 5-10 min at room temperature.
(Homogenate of Step 1 can be stored for 1 month at room temperature or for 10 months at 4°C or-20°C.)

Step 2．Centrifugation (optional) *2
1. In the case of tissues, sediment the homogenate for 10 min at 10,000 x g at 4°C or roomtemperature.
2. Following centrifugation, transfer the resulting viscous supernatant to a fresh tube. Takecare to avoid transferring any insoluble material.

*2 This step removes insoluble tissue fragments, RNA, and excess polysaccharides from thelysate/homogenate. It is required for the isolation of DNA from tissues such as liver, muscles
containing a large amount of cellular and/or extracellular material. This process is recommended inorder to minimize RNA carry-over into the DNA.

Step 3．DNA Precipitation
1. Precipitate DNA from the homogenate by the addition of 0.5 ml of 99.5% ethanol per 1 mlof ISOGENOME used for the isolation.
2. Mix samples and homogenate by inverting tubes 5-8 times.
3. Store at room temperature for 1-3 min.
4. DNA should quickly become visible as a cloudy precipitate. Transfer the DNA to a clean
tube by spooling with a pipette tip. Swirl the DNA onto the tip and attach it to the tube wallnear the top of the tube by gently sliding the DNA off the tip. *3*3 If no precipitate is visible, or if it is too dispersed to easily collect with a pipette tip, centrifuge for 5
min at 5,000 x g and recover the pellet.

Step 4．DNA Wash
1. Add 1 ml of 75% ethanol and gently mix by inverting the centrifuge tube 3-6 times.
2. Store the tubes vertically for 0.5-1 min, or centrifuge for 1-2 min at 1,000 x g, to allow theDNA to settle to the bottom of the tubes and remove ethanol by pipetting or decanting.
3. Wash the DNA precipitate again (repeat 1-2 of Step 4). (During washes, DNA can be stored
in 95% ethanol for at least one week at room temperature or for 3 months at 4°C.)

4Step 5．DNA Solubilization
1. Remove any remaining ethanol from the bottom of a tube using a pipette.
2. Dissolve DNA in 8 mM NaOH or ddH2O. *4
The DNA preparations isolated from tissues such as liver, muscles contain someinsoluble material (mostly polysaccharides). Remove the insoluble material bycentrifugation at 12,000 x g for 10 min.
3. DNA samples solubilized in 8 mM NaOH is neutralized with HEPES. *5*4 Add an adequate amount of 8 mM NaOH or ddH2O to approach a DNA concentration of 0.2-0.37µg/µl. Typically, add 0.2-0.3 ml of 8 mM NaOH or ddH2O to the DNA isolated from 10 cells or10-20 mg animal tissue.
5* For 1 ml of 8 mM NaOH use the following amounts of 0.1 or 1M HEPES (free acid).
＜Adjustment of pH in DNA samples solubilized in 8mM NaOH＞
Final pH

0.1 M HEPES

Final pH1 M HEPES

8.486 µl7.223 µl8.293 µl7.032 µl8.0101 µl

7.8117 µl

7.5159 µl

5＜Flowchart for Extraction of Genomic DNA＞
Sample (25-50 mg tissue, or up to 1×107 cells, either in pellet or in suspension (volume < 0.1 ml)
Add 1 ml ISOGENOME.
Homogenize by gently and thoroughly.
Centrifuge for 10 min at 10,000 x g.

(optional)

Supernatant (Transfer supernatant to a new tube)
Add 0.5 ml of 99.5% ethanol.
Mix samples and homogenate by inverting tubes 5-8 times.
Store at room temperature for 1-3 min.
Transfer the DNA to a clean tube by spooling with a pipette tip.
(Alternatively, collect the pellet by centrifugation for 5 min at 5,000 x g.)
the DNA pellet
Add 1 ml of 75% ethanol.
Mix by inverting the centrifuge tube 3-6 times.

Wash the DNA twice.

Store the tubes for 0.5-1 min and remove ethanol.
the DNA pellet (Remove as much of the supernatant as possible.)
Dissolve DNA in 8 mM NaOH or ddH2O.
the DNA sample (DNA samples solubilized in 8 mM NaOH is neutralized with HEPES.)

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＜Protocol B: DNA and RNA extraction combined with ISOGEN II＞
ISOGENOME can be isolated of DNA (for use in PCR) and RNA from same sample by theuse of combination with RNA extraction regent ISOGEN II (Code No. 311-07361).

Add 1 ml of ISOGEN II reagent to sample to homogenize
Add 0.4 volume water (RNase free)Mix by shaking for 15sec, incubate 5-15 min at room temperature
Centrifuge for 15 min at 12,000 x g

Supernatant

Precipitates

RNA can be recovered in procedures ISOGEN II.

DNA can be recovered in procedures ISOGENOME.

Please refer to the following.

Step 1．Lysis of Sample (Homogenization)
1. Remove any residual supernatant without disturbing the precipitate obtained after
centrifugation of the ISOGEN II containing homogenate.
2. Vigorously mix the pellet with 8 - 10 volumes of ISOGENOME and incubate for 5-10 min
at room temperature.

Perform steps 2 and later of Protocol A.

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VI

Rerated products
Code No.
318-08111
314-08113
317-07363
311-07361

Product Name

10 ml

ISOGENOME

100 ml
10 ml

ISOGEN II

100 ml

315-02504
317-02503

10 ml
ISOGEN50 ml

311-02501
317-02623
311-02621
318-01793
312-01791

100 ml
10 ml

ISOGEN-LS

100 ml0.02 mlEthachinmate

0.2 ml316-90101
318-90105

Size

100 mlDistilled Water, Deionized, Sterile

500 ml312-90103

100 ml×6314-90021

100 ml316-90025

500 mlTE（pH 8.0）

310-90023

100 ml×6

NIPPON GENE CO., LTD.
www.nippongene.com
If you have any questions, please contact us by web form.
The information in the descriptions of the products may be changed without prior notification.

ISOGENOME – Manual (Ver. 2) 1501HT-1805

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