1991-A Simple and Efficient Method For Isolation of DNA

6656 Nucleic Acids Research, Vol. 19, No.
23
A simple and efficient method for isolation of DNA

fragments from agarose gel
Tapas Mukhopadhyay1 and Jack A.Roth1l 2
Departments of 'Thoracic Surgery and 2Tumor Biology, The University of Texas M.D. Anderson
Cancer Center, 1515 Holcombe Boulveard, Box 109, Houston, TX 77030, USA
Submitted June 21, 1991
We describe a simple method for isolation of purified DNA ACKNOWLEDGEMENT

fragments from agarose gels. There are different methods for
isolation of DNA from gels, but the major disadvantage of most We wish to thank Carol Torrence in the preparation of this
of the techniques is that the recovery is often low for the manuscript. This study was partially supported by grants
fragments of longer or shorter sizes. Some methods are inefficient (CA45178) from the National Cancer Institutes of Health
at removing impurities that interfere with subsequent enzymatic
[J.A.R.]; Brown and Mathers Foundation [J.A.R.]; and by gifts
manipulation of the DNA. The method we describe is quick, to the Division of Surgery from Tenneco and Exxon for the core
efficient, and does not require expensive materials. It enables lab facility.
us to recover a high yield of intact pure DNA fragments of any
size.
The procedure involves the electrophoresis of the DNA
fragment in 1 % low-melting-point agarose (BRL) gel either in
TAE or TBE buffer. Sephadex G-50 beads were equilibrated with
TE buffer and autoclaved. A Sephadex G-50 column was made
in a 10-ml syringe and spun at 3000 rpm for 5 min; dried
Sephadex beads were stored in a tube. The bottom of a 1.5-mi
microfuge tube was plugged with a small amount of sterile
siliconized glass wool. The desired DNA band was excised from
the ethidium-bromide-stained gel. A volume of Sephadex G-50
dry beads was placed on top of the glass wool. The gel slice was
placed on top of Sephadex beads. The tube was closed and placed
in a 55°C to 60°C water bath to melt the gel (5 to 10 min, Figure 1. One microgram of Hindlll digested bluescript vector was size
approximately). As the gel slice melted, it entered into the fractionated in 1 % low-melting-point agarose gel at 80 V for 1 h (a). A linearized
Sephadex pack. A volume of TE buffer was added and incubated single DNA band was excised from the gel and eluted according to the method
described. The total eluted DNA was further run on the same type of gel and
for another 5 min at 55°C. The tube was then placed in ice for photographed (b).
5 min. Two small holes were made, one at the top and another
at the very bottom of the tube, with a 21-gauge needle. The tube
was placed on top of another microfuge tube and spun at 1000 Table I. Recovery of DNA fragments from agarose gels
rpm for 5 min at 4'C in a tabletop centrifuge. About 85 % of Gel Size Radioactivity Radioactivity eluted % Recovery
the DNA came out in the flowthrough. However, if 15 to 20 frag (103 bp) in vel slice from column
1d of TE buffer was added to the tube, further centrifugation (l0- Cpm) (Total) (l05 cpm) (Total)
would increase the yield to 95 %. Since the DNA fragment passes
1 23.1 1.97 (197910) 1.8 (186850) 94.4
through the Sephadex beads, it is clean and ready for subsequent 2 9.4 1.05 (105270) 0.95 (95753) 90.9
manipulation. This method is also applicable to standard agarose 3 6.6 0.75 (75706) 0.69 (69893) 92.3
gels, but the gel piece melts at 80°C, and 2 equivalent volumes 4 4.4 0.61 (61230) 0.57 (57930) 94.6
of TE buffer must be added during the melting of the gel. 5 2.3 0.60 (60975) 0.56 (56645) 92.8
To determine the percentage of recovery, restriction fragments 6 2.0 0.43 (43736) 0.41 (41716) 95.3
7 0.6 0.58 (58210) 0.55 (55985) 96.1
of different sizes of HindIII digested DNA (BRL) were nick
translated under mild conditions with ct-32p labeled dCTP. The The percent recovery of the DNA fragments which ranged from 500 bp to 20
DNA was run on 1 % low melting point agarose gel; ethidium- kb. Usually, about 80% to 90% recovery is achieved regardless of fragment size.
bromide-stained gel bands were excised and the radioactivity of However, it should be noted that high molecular weight DNA fragmients are more
the gel pieces was counted. The DNA from each gel piece was efficiently recovered. Frequently, high molecular weigh DNAs are diff-icult to
elute from the gel piece; either the recovery is very low or the DNA is broken
eluted through the column as described above and the percent down during extraction. This method allows an efficient alternative for isolating
radioactivity recovered was calculated as shown on Table 1. particularly large DNA fragments.

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6656 Nucleic Acids Research, Vol. 19, No.

A simple and efficient method for isolation of DNA

Submitted June 21, 1991

We describe a simple method for isolation of purified DNA ACKNOWLEDGEMENT

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