ISOSPIN Blood & Plasma DNA
Manual (Ver. 02)

Code No. 312-08131

NIPPON GENE CO., LTD.

IDescription

The ISOSPIN Blood & Plasma DNA enables a rapid isolation of highly pure DNA from blood,
serum and plasma.

II Kit components
Componet(50 preps)

Note

BE Buffer

15 ml x 1

Proteinase K

1 ml x 1

BW1 Buffer

48 ml x 1

(includes ethanol)*BW2 Buffer

35 ml x 1

(includes ethanol)*Elution Buffer

14 ml x 1

10 mM Tris-HCl (pH 9.0), 0.1 mM EDTA

Spin Column

50 sets x 1(a Spin Column and Collection Tube set)

Collection Tube50 tubes x 2*III

Keep the buffer bottles tightly closed after use.

Storage conditions

Proteinase K should be stored at -20°C. The remaining 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
・Ethanol・Micropipette・Pipette tips
・1.5 mL microcentrifuge tubes
・Microcentrifuge
・Vortex Mixer
・Water baths or heat blocks (at 56°C)

Material, as necessary
・Saline solution (Do not use buffer solution such as PBS.)
・Ribonuclease (DNase free) Glycerol Solution (Nippon Gene, Code No. 312-01931)

Before You Begin□ Warm blood samples to room temperature (15-25°C).
□ Set a water baths or heat block at 56°C.

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DNA Extraction Protocol1.

Add 20 µl of Proteinase K to a 1.5 mL microcentrifuge tube.

2.

Warm blood samples to room temperature (15-25°C), and mix well byinversion. Add 200-250 µl of the sample to the tube.
Note) If using <200 µl blood sample, adjust the sample volume to 200 µl using saline
solution. (Do not use buffer solution such as PBS. Using PBS may cause inefficient DNAextraction.)
Note) If using nucleated red blood (e.g., bird, reptile, amphibian or fish), add 190 µl of saline
solution to 10 µl of nucleated red blood (the sample will be 200 µl as a starting material.)

Opt. OPTIONAL STEP (A)a1)

If RNase treatment is required, add 20 µl of Ribonuclease (DNase free)
Glycerol Solution to the sample, and mix. Spin down lightly.

3.

Add 1 sample volume (200-250 µl) of BE Buffer to the tube, mix well byvortexing for 15 sec. Spin down lightly.

4.

Incubate at 56°C for 10 min.

5.

Add 1 sample volume (200-250 µl) of 96-100% Ethanol to the tube, mix
well by vortexing for 15 sec. Spin down lightly.
↓6.

Prepare a Spin Column (a Spin Column and Collection Tube set). Apply all
the mixture to the Spin Column.
Note) If using >200 µl serum or plasma sample, you can apply any remaining mixture to thesame Spin Column several times (up to 5 times) by repeating Step 6 and Step 7. But, to avoid
clogging is preferable to further addition.

7.

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

8.

Discard the flow-through with the Collection Tube. Place the Spin Column
into a clean Collection Tube.
↓3

9.

Add 750 µl of BW1 Buffer to the Spin Column.

10.

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

11.

Discard the flow-through with the Collection Tube. Place the Spin Column
into a clean Collection Tube.
↓12.

Add 500 µl of BW2 Buffer to the Spin Column.

13.

Centrifuge at 12,000 x g, for 5 min at room temperature.

14.

Discard the flow-through with the Collection Tube. Transfer the Spin Column
to a new 1.5 mL microcentrifuge tube.
Note) Avoid contact of the Spin Column with the flow-through. If in doubt, perform next
Optional Step (B).

↓Opt. OPTIONAL STEP (B)b1)

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

b2)Discard the flow-through with the 1.5 mL microcentrifuge tube. Transfer theSpin Column to a new 1.5 mL microcentrifuge tube.

↓15.

Add 20-200 µl of Elution Buffer to the center of the Spin Column. Incubate
3 min at room temperature.
Note) Nuclease-free water can also be used to elute the DNA in place of Elution Buffer (10mM Tris-HCl, 0.1 mM EDTA, pH 9.0). We recommend using Elution Buffer containing EDTA if
you store the eluted DNA for long-term storage.
Note) Larger elution volumes can increase yield of DNA at the cost of dilution of the sample(see the page 7.)

16.

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

17.

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

4Simplified ProtocolWarm blood (serum or plasma) samples to room temperature (15-25°C).
Add 20 µl of Proteinase K to a 1.5 mL microcentrifuge tube.
Mix blood sample by inversion. Add 200-250 µl of the sample to the tube.
Add 20 µl of Ribonuclease (DNase free) Glycerol Solution to the sample. [Opitional Step]

Add 1 sample volume (200-250 µl) of BE Buffer to the tube,
mix well by vortexing for 15 sec. Spin down lightly.
Incubate at 56°C for 10 min.
Add 1 sample volume (200-250 µl) of 96-100% Ethanol to the tube,
mix well by vortexing for 15 sec. Spin down lightly.
Prepare a Spin Column. Apply all the mixture to the Spin Column.
Centrifuge at 12,000 x g, for 1min at room temperature.
Discard the flow-through with the Collection Tube.
Place the Spin Column into a clean Collection Tube.
Add 750 µl of BW1 Buffer to the Spin Column.
Centrifuge at 12,000 x g, for 1 min at room temperature.
Discard the flow-through with the Collection Tube.
Place the Spin Column into a clean Collection Tube.
Add 500 µl of BW2 Buffer to the Spin Column.
Centrifuge at 12,000 x g, for 5 min at room temperature.
Discard the flow-through with the Collection Tube.
Transfer the Spin Column to a new 1.5 mL microcentrifuge tube.
Centrifuge at 12,000 x g, for 1 min at room temperature to remove residual liquid.
Transfer the Spin Column to a new 1.5 mL microcentrifuge tube. [Optional Step]
Add 20-200 µl of Elution Buffer to the center of the Spin Column.
Incubate 3 min at room temperature.
Centrifuge at 12,000 x g, for 1min at room temperature.
Recover your purified DNA in the microcentrifuge tube.
5VI

Troubleshooting

Low DNA yield
When adding BE Buffer, mix well by vortexing for more than 15 sec.
When adding 96-100% Ethanol, mix well by vortexing for 15 sec.
For increased DNA yield, add 100-200 µl of Elution Buffer in Step 15.
Do not use distilled water less than or equal to pH7.0, instead of Elution Buffer in Step 15.
Discolored membrane after washing with BW1 Buffer
When adding BE Buffer, mix well by vortexing for more than 15 sec.
Wash again with BW1 Buffer (repeat the Steps 8-11, before the Step 12.)Low concentration of DNA
For higher concentration, add 20-50 µl of Elution Buffer in Step 15. Smaller elution volumes
can increase concentration of DNA at the cost of yield of the sample.
Low DNA Performance
Perform the Optional Step (B) to remove BW2 Buffer from the Spin Column.

6VII Data
The DNA yield and concentration
varies with Elution Buffer volume.
Yield and concentration using different
elution volumes are expressed as apercentage of the sample using a 100 µl
elution volume.
For each Elution volume, the relative
yield (bar chart)

and the relative

concentration (line chart) are depictedin this data.

The DNA yield varies with whiteblood cell count.
Genomic DNA was purified from 100 µl
human whole blood with the kit using a
100 µl Elution Buffer.

VIIIRerated products

Code No.

Product Name

Size

312-01931

Ribonuclease (DNase free ) Glycerol Solution

1 ml

319-08141
315-08143

Collection Tube

100 tubes
500 tubes

NIPPON GENE CO., LTD.
URL: http://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.
ISOSPIN Blood & Plasma DNA Manual (Ver. 02) 201501-1704

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