Millipore - DNA Extraction from Agarose Gels with Montage Gel Extraction Kit or Ultrafree-DA Centrifugal Filters

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DNA Extraction from Agarose Gels with Montage Gel Extraction Kit or Ultrafree-DA Centrifugal Filters

Introduction

The Ultrafree-DA device is designed to recover 100 to 10,000 bp DNA from agarose gel slices in one 10-minute spin. It consists of a pre-assembled sample filter cup with an agarose gel nebulizer and a microcentrifuge vial. The device uses gel compression to extract DNA from the agarose. Centrifugal force collapses the gel structure, drives the agarose through a small orifice in the gel nebulizer and captures the resultant gel slurry in the sample filter cup. As the agarose is compressed at 5,000 x g, DNA is extruded from the gel's pores. The gel matrix is retained by the microporous membrane, and the DNA passes freely through the membrane. DNA can then be recovered in the filtrate vial.

The Montage Gel Extraction Kit consists of 50 Ultrafree-DA centrifugal filters as well as a modified TAE buffer that allows the casting and running of the gel from which the DNA fragment is to be extracted.

DNA prepared with the Ultrafree-DA centrifugal filter requires no further purification for most applications, including cloning and radioisotopic or fluorescent DNA sequencing. Since agarose gel electrophoresis has high resolving power, the small and large non-specific amplification products that frequently interfere with cloning and sequencing after PCR (polymerase chain reaction) are completely removed from the product.

Materials

Microcentrifuge
Pre-assembled Ultrafree-DA centrifugal filter device or Montage Gel Extraction Kit
Modified TAE* electrophoresis buffer (40 mM Tris-acetate, pH 8.0, 0.1 mM Na2EDTA)
SeaKem agarose (FMC BioProducts) or equivalent
Long-wavelength UV lamp
Scalpel or razor blade

Procedure

Electrophorese 30 µL of PCR product or other DNA through a <1.25% ordinary agarose gel, prepared in modified TAE buffer with ethidium bromide (0.5 µg/mL).
Locate the band of interest with a long wavelength UV lamp or transilluminator. With a razor blade or scalpel, cut out the slice of agarose (<100 µL or 100 mg) containing the band of interest. Trim any excess agarose away from band.
Place the gel slice into the gel nebulizer/sample filter cup/filtrate vial assembly and seal the device with the cap attached to vial.
Spin at 5,000 x g for 10 minutes. Centrifugation forces the agarose through the gel nebulizer, converting it to a fine slurry that is captured by the sample filter cup. Extruded DNA in electrophoresis buffer passes through the microporous membrane in the sample filter cup and collects in the filtrate vial.
DNA in the filtrate is now ready for sequencing or cloning without further purification. Discard the gel nebulizer and sample filter cup and store the DNA in the capped filtrate vial.

* Modified TAE is recommended rather than TBE for the following reasons: (1) TBE buffer strongly inhibits DNA sequencing reactions while modified TAE buffer does not. (2) Modified TAE has 0.1 mM Na₂EDTA while regular TAE has 1.0 mM Na₂EDTA. The EDTA level at 0. 1 mM Na₂EDTA will not interfere with the magnesium concentration in sequencing reactions and other downstream enzymatic treatments, many of which are dependent on magnesium.

Results

Gel compression is a quick and easy technique for recovering DNA from an agarose gel slice. Table 1.1 Effect of gel disruption on typical DNA recoveries from agarose gels

DNA Size (bp)	% DNA Recovered from Intact Gel	% DNA Recovered from Gel Disrupted by Gel Nebulizer
100	74	78
400	39	ND
700	43	71
1000	55	77
2027	*	47
4361	14	35
9416	*	32
23130	*	29
* = Not detectable

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