ANALYTICAL BIOCHEMISTRY 169, 194-196 (1988)

Simultaneous Electroelution and Concentration of

DNA Fragments from Agarose Gels

WILLIAMG.STROOP
Neurovirology Research Laboratory. I ‘eterans .4dministraiion Medical Center: Departments of Nerrrolc),qy and
Pathology. University of’ Utah Medical School, Salr Lake C’it!: (ilah 54148

Received July 27. 1987

A rapid and inexpensive method for the electroelution of DNA fragments from agarose gels is
described. DNA fragments were separated by agarose gel electrophoresis and visualized hy
staining with ethidium bromide. Selected DNA fragments were placed into electroeluter tubes
capped with dialysis membrane and electroeluted into a small volume of buffer using a con-
ventional horizontal gel electrophoresis apparatus. The method successfully eluted and con-
centrated DNA fragments with molecular weights ranging from 2.7 to 13.9 MDa in 3 h. C: 19x8
Academic Press, Inc.
KEY WORDS: DNA: electrophoresis; cloning; sequencing.

Agarose gel electrophoresis of DNA frag-
ments produced by restriction endonuclease
digestion is a common technique employed
in molecular biology. DNA fragments recov-
ered from agarose gels are used for many
subsequent experiments, and many methods
have been developed to recover DNA frag-
ments from gels (l-3). Electroelution
DNA fragments from agarose gels is one of
the frequently employed procedures for re-
covering restriction-enzyme-digested DNA
from agarose gels and involves the elution of
the DNA into a buffer solution by applica-
tion of an electric current. Several relatively
expensive electroelution apparatuses
available commercially. This report de-
scribes a simple, rapid, and inexpensive tech-
nique for electroelution which utilizes a
horizontal agarose electrophoresis ccl 1 pre-
eluding the need to purchase addii :ifonal
equipment.
MATERIALS AND
DNAs from chimeric plasmids p! 3 3.
pSG 16, pSG 17, pSG 22, and pSG 28
containing herpes simplex virus, type 1
(HSV-I),’ strain KOS genomic DNA inserts
of3.4, 10.4, 2.7, 8.6, and 13.9 MDa, respec-
tively (4) were purified from Escherichia c&i
HB 101 cells by the methods of Birnboim and
Doly (5). Approximately 100 ng of plasmid
DNAs was digested to completion with
EcoRI (E. co/i RY 13) (International Bio-
of technologies, Inc., New Haven, CT) for 4 h
at 37°C under manufacturer-recommended
conditions. Digestion was stopped by addi-
tion of 20 ~1 of 50 mrvr EDTA, and the reac-
tion volume was reduced to 10 ~1 using a
Savant Speed-Vat Concentrator (Savant In-
struments. Inc., Hicksville, NY). Electropho-
are resiswas performed for 17 h at 45 V constant
using an International Biotechnologies
Model MPH unit and l-cm-thick 0.7% aga-
rose gels in Tris-acetate buffer (6). Digested
pSG DNAs were visualized by ethidium bro-
mide staining (Fig. 2) and the HSV-1 KOS
inserts were excised from the gel with a scal-
pel for electroelution. Electroeluter tubes
METHODS were prepared as shown in Fig. I. After elec-
troelution at 125 V constant for 3 h, the cur-
’ Abhreviation used: HSV-I. herpes simplex virus,
type I.

0003-2697/88 $3.00 194

Copyright IC’ 19X8 by Academnc Press. Inc.
All rights of repruductlon I” any form nerved
 ELECTROELUTION OF DNA 195

o- f
DNA. Electroelution of the KOS DNAs was
rapid. in that the bromphenol blue tracking
dye passed through the agarose matrix and
the dialysis membrane within 30 min. Re-
covery of electroeluted DNA was confirmed
by electrophoresis (Fig. 2). Recovery of DNA
was roughly proportional to its molecular
Electrophoresis Cell 4
weight, although the process was more suc-
I I
Buffer
cessful with inserts from pSG 16. pSG 33.
and pSG 78 with molecular weights of 10.4,
Buffer Buffer
u’ 8.6. and 13.9 MDa, respectively, than dialy-
sis-bag (7) and slot electroelution (2)
1-l (+I
methods. By comparison of the intensities of
FIG. 1. Construction of the electroeluter. Nine-milli- ethidium-stained DNA samples of known
meter (0.d.) glass tubing was bent to fit into the electro- concentration in adjacent lanes of separate
phoresis cell. Oriented as shown, the elbow of the tube gels (8.9). it was estimated that 60-70% re-
was filled with 0.7% agarose (a) which was quick hard- covery of 13.9 MDa DNA. 70-80% of 10.4
ened in an ice bath. Ten microliters ofsample buffer w’as
MDa DNA, and 80-90% recovery of 1.7
added (b). and the agarose plug containing the DNA
sample was inserted with forceps and anchored in place
MDa DN.4 were achieved. With the DNAs
by addition of a small amount of 0.7’;’ agarosc (c). The
dead space at the end of the tube (d) was filled with II
f oh I I
electrophoresis buffer. and the tube was capped with a
boiled and EDTA-treated dialysis membrane with a
12.000- to 14.000-Da molecular weight cutolf (e). The
dialysis membrane was held in place with a 4-mm (i.d.)
O-ring (f). The remainder of the tube was filled with 13.8-
buffer. and the tube was placed into the electrophoresis
cell containing buffer as shown.

3.7 -
3.6-
rent was reversed for 2 min. and the electro- 3.2 -

eluted DNA was extracted with phenol, 4.8

phenol:chloroform, and chloroform. precipi- 2.3-
3.7
tated in sodium acetate/ethanol, dried, and 3.6
3.2
resuspended in 10 mM Tris, pH 7.4, and 1
mM EDTA. Agarose was removed from the
tubes by heating them in a microwave, and 2.3
the tubes were subsequently cleaned, auto-
claved, and reused. FIG. 2. (I) Agarose electrophoresis of Ec.oRI-digested
pBR375 DNAs containing HSV- I (strain KOS) genomic
inserts in plasmids pSG 3 (lane a). pSG 16 (lane b). pSG
RESULTS AND DISCUSSION 17 (lane c). pSG 22 (lane d). and pSG 28 (lane c). (II)
Agarose electrophoresis of inserts electroeluted from
The method described here resulted in pSG 3 (lane f), pSG I6 (lane g). pSG 17 (lane h), pSG 27
concentration of the electroeluted DNA into (lane i). and pSG 28 (lane j). Note that the insert from
a very small amount of buffer which was ad- pSG 3 (lane f) is free of contaminating pBR335 DNA.
The clectrophoresis time for gel II was longer than for gel
vantageous for the subsequent phenol ex-
I to better assess purity and molecular weights of the
traction stepsusedto separate the DNA from insert DNAs. Lines indicate the position of X-DNA
high molecular weight sulfated polysaccha- EuTRI fragments of known molecular weights (MDa)
rides eluted from the agarosealong with the coelectrophoresed in an adjacent lane ofeach gel.
196 WILLIAM G. STROOP

used, no advantage was found to using 2. Yang. R. (‘.-A.. Lts. J., and Wu. R. (lY7Y) r!!
higher voltages or longer times for electroelu- Methods in Enzymology (Wu. R.. Ed.). Vol. 6X.
pp. 176-I X3. 4cademic Press. New \‘ork.
tion. However, it is suspected that longer
3. Wu. R., Jay. F.. and Roychoudhury. R. (1976)
and/or higher voltages times may be required .zfl~lllcdv (‘llrrwr Rl,.\. 12, X7- 176.
to maximize recovery of fragments larger 3. Goldin. A. L.. Sandri-Golden. R. M., I.evine. M..
than 14-15 MDa. and Glorioso. J c‘. (I YX I ) .I 1 Irol 38, 50-5X.
5. Birnhoim. JI. C‘.. and Daly. J. t lY7Y) i\:ll(,/clc.. lc,rrlv
ACKNOWLEDGMENTS Rl,\ 7, 1513-1523.
This work was supported by the Veterans Administra- 6. Maniatis. ‘I ,. Frttsch. E. F.. and Samhrook. J. ( lYX7)
tion Service. We gratefully thank Dr. Myron Levine for Molecular C‘loning. pp. 150-l 72. Cold Spring
prov,iding the chimeric plasmids. Dr. R. E. Lynch for Harhor 1.ahoratot-y. Cold Spring Harbor, N\‘.
helpful discussions. and Douglas C. Schaefer for techni- McDonell. M. W.. Simon, M. N.. and Studier, F. W.
cal assistance. (lY77)./ \/(I/ H/o/. I IO, 119-146.
Sharp. P. A.. Sugden. B., and Samhrook. J. (1971)
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(Grossman. L.. and Moldave. K.. Eds.). Vol. 65. Methods in Molecular Biology. pp. 90-97.
Part I. pp. 37 l-380. Academic Press. New York. Springer-Verlag. New York