ISOSPIN Agarose Gel
Manual (Ver. 04)

Code No. 311-07981

NIPPON GENE CO., LTD.

IDescription

The ISOSPIN Agarose Gel enables an easy, rapid and effective isolation of DNA fragmentsfrom agarose gels for 30 min. The kit is applicable for DNA isolation from up to 5% standard
or low-melt agarose gels in TAE or TBE buffer.

II Kit components
Component(100 preps)

Note

ISAE Buffer

75 ml x 2

Yellow *ISW Buffer

100 ml x 1(includes ethanol) **ISE Buffer

10 ml x 1

10 mM Tris-HCl (pH 8.5)

Spin Column (a Spin Column and Collection Tube set)

50 sets x 2*If precipitates appear in the ISAE Buffer, please incubate at 37-60°C, inverting periodically to dissolve.

**Keep the buffer bottle tightly closed after use.

IIIStorage conditions

All the kit components can be stored at room temperature (15-25°C).

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IV Precautions
・ The kit is intended research use only.

V Protocol
Material not supplied
・Micropipette・Pipette tips
・1.5 mL microcentrifuge tubes
・Microcentrifuge
・Heater (at 50°C)
・Isopropanol

Material, as necessary
・2.0 mL microcentrifuge tubes
・3M Sodium Acetate (pH 5.2)

2Protocol
1.

Excise DNA fragment out of the gel with a clean scalpel and transfer it to aclean 1.5-2 mL microcentrifuge tube. Weigh the gel slice.
Note)・The gel slice should be ≤250 mg in a 1.5 mL microcentrifuge tube.
・The gel slice should be ≤340 mg in a 2.0 mL microcentrifuge tube.
・UV light Exposure time should be kept as short as possible.

↓2.

Add 3 volumes of ISAE Buffer (Yellow) to 1 volume of gel slice.
Example) Add 300 µl of ISAE Buffer to 100 mg of gel.

3.

Incubate at 50°C for 10 min, and mix by inverting the tube every 2 minduring the incubation until the gel slice has completely dissolved.

4.

After the gel slice has completely dissolved, check that the color of themixture is yellow.
Note) If the color of the mixture becomes orange or purple, add 1/60 volumes of 3M Sodium
Acetate (pH 5.2) to 1 volume of the mixture. The color will return yellow.
e.g., Add 6.7 µl of 3M Sodium Acetate (pH 5.2) to about 400 µl of the mixture (100 mg of thegel and 300 µl of the ISAE Buffer).

5.

Add 1 volume of isopropanol to 1 gel volume (i.e., add 1/4 volumes ofisopropanol to 1 volume of the mixture), and mix well by inverting. Spin down
lightly.
Example) Add 100 µl of isopropanol to about 400 µl of the mixture (100 mg of the gel and 300µl of the ISAE Buffer).

↓
6.

Prepare a Spin Column (a Spin Column and Collection Tube set). Apply theMixture to the Spin Column.

7.

Centrifuge for 1 min at 12,000 x g at room temperature.

8.

Discard the flow-through. Place the Spin Column back into the same
Collection Tube.
Note) If there are the Mixture left over, apply the remaining Mixture to the Spin Column, andrepeat 7 and 8.

↓3

9.

Apply 500 µl of ISAE Buffer (Yellow) to the Spin Column.

10.

Centrifuge for 1 min at 12,000 x g at room temperature.

11.

Discard the flow-through. Place the Spin Column back into the same
Collection Tube.
↓12.

Apply 750 µl of ISW Buffer to the Spin Column.

13.

Centrifuge for 1 min at 12,000 x g at room temperature.

14.

Discard the flow-through. Place the Spin Column back into the sameCollection Tube.
↓15. Centrifuge for 1 min at 12,000 x g at room temperature to remove residualliquid.

↓16.

Transfer the Spin Column to a new 1.5 mL microcentrifuge tube.

17.

Add 50 µl of ISE Buffer to the center of the Spin Column. Incubate 3 min atroom temperature.
Note) Nuclease-free water or TE (pH8.0) can also be used to elute the DNA in place of ISE
Buffer (10 mM Tris-HCl, pH 8.5).

18.

Centrifuge for 1 min at 12,000 x g at room temperature.

19.

Recover your purified DNA fragments in the microcentrifuge tube.
Note) The DNA fragments can be used directly or stored at -20°C for long term storage.

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Simplified Protocol
Excise gel slice and transfer it to a 1.5-2 mL microcentrifuge tube.
Add 3 volumes of ISAE Buffer (Yellow) to 1 volume of the gel slice.
Incubate at 50°C for 10 min, and mix by inverting the tube every 2 minduring the incubation until the gel slice has completely dissolved.
［If the color of the mixture becomes orange or purple,
add 1/60 volumes of 3M Sodium Acetate (pH 5.2) to 1 volume of the mixture.］
Add 1/4 volumes of isopropanol to 1 volume of the mixture.
Mix well by inverting.
Spin down lightly.

Apply the Mixture to a Spin Column.
Centrifuge for 1 min at 12,000 x g at room temperature.
Discard the flow-through.
Add 500 µl of ISAE Buffer (Yellow).
Centrifuge for 1 min at 12,000 x g at room temperature.
Discard the flow-through.
Add 750 µl of ISW Buffer.
Centrifuge for 1 min at 12,000 x g at room temperature.
Discard the flow-through.
Centrifuge for 1 min at 12,000 x g at room temperature to remove residual liquid.
Transfer the Spin Column to a new 1.5 mL microcentrifuge tube.
Apply 50 µl of ISE Buffer to the center of the Spin Column.
Incubate 3 min at room temperature.
Centrifuge for 1 min at 12,000 x g at room temperature.
Recover your purified DNA fragments in the microcentrifuge tube.

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VI

Troubleshooting

Low DNA Yield
Ensure the ISE Buffer is applied to the center of the Spin Column so that elution is efficient.
Larger elution volumes can increase yield of DNA at the cost of dilution of the sample.
Ensure reagents will be added correctly.
Be sure to incubate until the gel slice has completely dissolved.
The ISAE Buffer (Yellow) is acidic. If the pH increases during the gel melting process, thecolor of the mixture will become Orange or Purple. In such case, add 3M Sodium Acetate (pH5.0) to adjust the pH of the mixture.

Low DNA Performance
Please repeat wash step (Step 12, 13 and 14). 80% Ethanol can also be used to wash theDNA in place of ISW Buffer.
If ethanol has been carried-over, spin for 5 min, instead of 1 min in Step 15.

Sheared DNAMix well by inverting or flicking the tube. Do not mix by pipetting.

6VIIData

Technical Information
DNA Binding Volume

20 µg

Column Volume

900 µlMinimum Elution Volume:

10 µl

Typical DNA Recovery

40 – 80% (100 bp – 20 kbp)

Typical recoveries with smaller and larger fragment sizes

The information in the descriptions of the products may be changed without prior notification.

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

http://www.nippongene.com/ISOSPIN Agarose Gel Manual (Ver. 04) 201311-1704

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