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CAT. NO.: mB003
Package: 100 Preps
Components
Spin Column & Collection Tube
Buffer B2
Wash Solution
Elution Buffer
Protocol mB003, 100 Preps
100 sets
60 ml
24 ml
10 ml
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Transportation at ambient temperature, store at room temperature.
Buffer B2 contains guanidine salt, thus is irritating to eyes and skin, please avoid contact with eyes, skin, and clothes.
Wash thoroughly after handling. Keep container closed.
24-hour emergency information
Emergency medical information in English, French, and German can be obtained 24 hours a day from: Poison
Information Center Mainz, Germany. Tel.: +49-(0)6131-19240
miniBio Column DNA Gel Extraction Kit utilizes silica-gel based membrane, which selectively absorbs DNA fragments up to 10 µg in the presence of specialized binding buffers after liberation from agarose gel. DNA fragments can then be eluted readily with Elution Buffer.
1. Rapid and economical.
2. High yields (60-90% recovery). It is suitable to recover 60 bp - 10 kb DNA fragments.
3. Efficient removal of contaminants. Purified DNA can be used in most downstream applications such as sequencing, labeling, restriction enzymatic digestion, ligation or transformation.
4. No phenol/chloroform extraction or ethanol precipitation.
Microcentrifuge capable of at least 12,000 × g
Sterile 1.5 ml or 2 ml Centrifuge Tubes
Absolute ethanol (96%-100%)
Updated:07/2015 www.minibio.de

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Before use, add 96 ml absolute ethanol to the 24 ml Wash Solution (volume of ethanol:volume of Wash Solution=4:1).
Elution Buffer is 2.0 mM Tris-HCl pH 8.5. Although TE buffer pH 8.0 or water can be used, yield is generally 20% lower.
Check the Buffer B2 for salt precipitation before each use. If necessary, redissolve the precipitate by warming the solution at 37°C, then cool back down to room temperature before use.

All centrifugations are carried out at 13,000 rpm (~17,900 x g) with a conventional, table-top microcentrifuge.

All steps should be carried out at room temperature (15 –25°C).
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Weigh the gel slice and transfer to a 1.5 ml microfuge tube.
Note: Try best to incise the agarose gel as small as possible. If the weight of the gel slice is less than 100 mg, add ddH
2
O to
100 mg.
2. Add Buffer B2 to agarose gel cut-out (check table below for reference). Incubate at 5060ºC for 10 min and shake occasionally until agarose is completely dissolved.
% Agarose gel < 1 1-1.5 1.5-2 >2
μl Buffer B2 per 100 mg cut-out 200 300 400 500
Note: For DNA fragment <500 bp or >4 kb, please add 100 μl isopropanol per 100 mg of agarose gel in order to increase the yield. Make sure that the gel is completely dissolved before proceeding.
3. Transfer the above mixture solution to a clean spin column assembled in a 2 ml collection tube (provided) and spin for 30 sec.
Note: The maximum volume of the column reservoir is 75 0 µl. For sample volumes of more than 750 µl, simply load and spin again.
4. Discard flow-through and place the column back to the same collection tube.
Add 500 μl Wash Solution
(diluted with ethanol previously) to the column and centrifuge for 30 sec.
Note: Add absolute ethanol according to the bottle label to Wash Solution before use.
5. Repeat step 4 once.
6. Discard flow-through, place the column back to the same collection tube and centrifuge for 1 min.
Note: This step is important for removing residual ethanol, which may inhibit subsequent enzymatic reactions. Keep the column at room temperature for 5-10 minutes after this step can further remove the ethanol.
7. Place the column in a new clean 1.5-ml microfuge tube. Add 30-
40 μl of Elution Buffer to the center of the membrane and incubate for 1 minute. Spin for 1 minute to elute DNA. Store the DNA at 20ºC.
Note: In order to improve DNA elution efficiency, the elution buffer or ddH
2
O can be heated to 60ºC prior to apply to the column.
Updated:07/2015 www.minibio.de

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----------Detach this page to have a Short Protocol for Experienced User ----------

All centrifugations are carried out at 13,000 rpm (~17,900 x g) with a conventional, table-top microcentrifuge.

All steps should be carried out at room temperature (15 –25°C).
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Weigh the gel slice and transfer to a 1.5 ml microfuge tube.
2. Add Buffer B2 to agarose gel cut-out (check table below for reference). Incubate at 50-
60ºC for
10 min and shake occasionally until agarose is completely dissolved.
% Agarose gel
μl Buffer B2 per 100 mg cut-out
< 1
200
1-1.5
300
1.5-2
400
>2
500
3. Transfer the above mixture solution to a Spin Column placed in a 2 ml collection tube and centrifuge for 30 sec .
Note: For sample volumes of more than 750 µl, simply load and spin again.
4. Discard flow-through and place the column back to the same collection tube. Add 500 μl of Wash Solution (diluted with ethanol previously) , centrifuge for 30 sec .
5. Repeat step 4 once.
6. Discard flow-through, place the column back to the same collection tube and centrifuge for 1 min to remove residual ethanol.
7. Place the column in a new clean 1.5-ml microfuge tube. Add 3040 μl Elution Buffer to the center of the membrane of the column and incubate for 1 min . Centrifuge for 1 min to elute DNA and store at -
20ºC.
Updated:07/2015 www.minibio.de