Universal DNA Purification Kit

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Application
DNA Purification (DNA Purif)
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Purpose For the extraction of DNA fragments from agarose gels, PCR and enzymatic reactions.
Characteristics The Universal DNA Purification Kit is designed for efficient and convenient purification of DNA fragments from agarose gels, PCR and enzymatic reactions. The kit utilizes a silica-based membrane system in the form of a spin column, which can bind up to 10 μg of DNA selectively. Special buffers are optimized for efficient recovery of DNA and removal of proteins, salts, and organic molecules. The kit can be used to purify DNA fragments from 100 bp-10 kb in size, and recovery rates are 60 % -80 % in this size rage. DNA purified with Universal DNA Purification Kit is suitable for many subsequent applications, such as restriction, ligation, PCR, sequencing, labeling, in vitro transcription and microinjection.
Components Buffer PD, Buffer PW, Buffer EB, Spin Columns, Collection Tubes
Application Notes Optimal working dilution should be determined by the investigator.
Protocol DNA extraction from agarose gels

1. Excise the DNA fragment from an agarose gel. Remove extra agarose.
2. Weigh the gel slice in a clean tube. Add 3 times volumes of Buffer PD to 1 volume of gel (100 mg-100 μL). Incubate at 65 °C for 10 min. Vortex the sample briefly every 2-3 mins until the gel slice is completely dissolved.
For example, add 300 μL Buffer PD to each 100 mg of gel. For gels containing >2 % agarose, add 6 volumes of Buffer PD.
3. After the gel slice has dissolved completely, check that the color of the mixture is yellow, similar to Buffer PD without gel slice.
If the color of the mixture is pink or violet, add 10-20 μL 3 M sodium acetate pH 5.0, and mix. The color of the mixture will turn yellow.
When the DNA fragment <500 bp, add same volume of isopropanol with the gel to increase the yield of the DNA.
4. Place a spin column into a provided collection tube (2 ml) and load up to 750 μL sample. Centrifuge for 1 min at 12,000xg. Discard flow-through and place the column back to the collection tube.
For sample volumes of more than 750 μL, simply load and centrifuge again.
5. Add 700 μL buffer PW to the spin column. Centrifuge at 12,000xg for 1 min. Discard flow-through and place the column back to the collection tube.
6. Repeat step 5.
7. Centrifuge the spin column for an additional 1 min at 12,000xg to remove Buffer PW.
Make sure that the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
8. Place the spin column into a clean 1.5 mL microcentrifuge tube (not provided). Open the lid, and stand in the air for 1 min to evaporate the liquid. To elute DNA, add 50 μL of Buffer EB or deionized water ( pH 7.0-8.5) to the center of the membrane and incubate at room temperature (15-25 °C) for 1 min. Centrifuge at 12,000xg for 1 min.
Pre-warming the Buffer EB at 60 °C will generally improve elution efficiency.
For increased DNA concentration, add 30 μL of Buffer EB to the center of the membrane.

DNA extraction from PCR and other enzymatic reactions

1. Mix the sample with 5 times volumes of Buffer PD.
For example, mix 100 μL PCR reaction and 500 μL Buffer EB.
For very small sample volumes <100 μL adjust the volume of the reaction mixture with water.
When the DNA fragment <500 bp, add same volume of isopropanol with the gel to increase the yield of the DNA.
2. Place a spin column into a provided collection tube (2 ml) and load up to 750 μL sample. Centrifuge at 12,000xg for 1 min. Discard flow-through and place the column back to the collection tube.
For sample volumes of more than 750 μL, simply load and centrifuge again.
3. Continue with Step 5 of the protocol ""DNA extraction from agarose gels"".
Restrictions For Research Use only
Storage RT,4 °C,37 ºC
Storage Comment The Universal DNA Purification Kit can be stored for up to 12 months at room temperature (15-25°C) or at 2-8°C for storage periods longer than 12 months. Check buffers for precipitate before use and redissolve at 37°C if necessary.