ANALYTICAL

BIOCHEMISTRY
Analytical Biochemistry 321 (2003) 135137
www.elsevier.com/locate/yabio

Notes & Tips

DNA purication on homemade silica spin-columns

Tatiana A. Borodina, Hans Lehrach, and Aleksey V. Soldatov*
Max-Planck-Institut fur Molekulare Genetik, Abteilung Lehrach, Ihnestrasse 73, 14195 BerlinDahlem, Germany
Received 1 April 2003

We describe here the preparation and use of inex-
pensive homemade spin columns which are at least as
ecient in purication of DNA as commercially avail-
able kits. The minimal elution volume of homemade
columns is considerably smaller. The plasmid DNA
purication protocol proposed here does not involve the
use of RNase, which is important for in vitro tran-
scription experiments.
Both homemade and recharged columns can sub-
stitute for commercial columns in QIAprep and QIA-
quick kits (results not shown). DNA binding capacity is
about 16 lg (
8 lg) for 2(1) GF/F membranes. Because
of the compact geometry of the homemade columns
DNA may be eluted in a relatively small volume of
elution buer (Fig. 1B).

Column preparation
Silica membranes were cut out from GF/F borosili-
cate glass ber paper (Whatman, England) by a 7-mm
wad punch. GF/F has the largest DNA-binding capacity
(2.8 lg DNA/mg of membrane) compared to other types
of membranes: GF/A (0.10 lg/mg), GF/B (0.14 g/mg),
GF/C (0.46 lg/mg), and GF/D (0.13 lg/mg) (results not
shown).
Homemade columns. Sequential steps of column
preparation are shown in Fig. 1A. The cap of a 0.5-ml
microcentrifuge tube was cut o, but a small tail was
left. Several holes were punctured in the bottom of the
tube by a red-hot needle. One or two membranes were
inserted tightly into the tube. For loading and washing
the column was placed in a reusable 2-ml screw-capped
microcentrifuge tube; for DNA elution it was placed in a
1.5-ml tube.
Recharging of used commercial columns (Qiagen).
The used membrane and plastic gasket ring were pushed
out from the column by an unfolded paper clip. The
plastic parts were washed by detergent and water. To
prevent cross-contamination in sensitive applications,
they can be treated with a 5% solution of sodium hy-
pochlorite [1]. Parts were dried; new membranes were
inserted and xed by the gasket ring.
*
Corresponding author. Fax: +49-30-8413-1128.
E-mail address: soldatov@molgen.mpg.de (A.V. Soldatov).
Buer solutions
All solutions are stable for at least 4 months at room
temperature: plasmid buer (PB1),1 50 mM glucose,
35 mM EDTA, pH 8.0; PB2, 0.2 N NaOH, 1% SDS;
PB3 (should be prepared immediately before use from
two stable components), four volumes [6.625 M guani-
dine hydrochloride (GuHCl), 62.5 mM 2-(N-morpho-
lino)ethanesulfonic acid (MES), pH in the interval 56,
0.3 M acetic acid, 25% (v/v) ethanol] and one volume
[0.5 M EDTA, pH 8.0] (the nal mixture is stable for at
least 1 day); PB4, 5 M guanidine thiocyanate (GuSCN),
100 mM EDTA, pH 8.0; wash buer (WB), 10 mM Tris
Cl, pH 7.5, 80% (v/v) ethanol; elution buer (EB),
10 mM TrisCl, pH 8.5; adsorption buer (AB), 5 M
GuSCN, 50 mM MES, pH 56, 20 mM EDTA, pH 8.0,
0.5% Triton X-100, 0.05 mM cresol red; NaI buer
(NIB) 6.6 M NaI, 16 mM Na2 SO3 , 0.05 mM cresol red
(store at 4 C in the dark) magnesium buer (MgB), 5 M
GuSCN, 100 mM MES, pH 56, 250 mM MgCl2 , 0.5%
Triton X-100.
Cresol red is included in AB and NIB buers as a pH
indicator dye [2]. It is yellow at pH 6 7.5, which is op-
timal for the DNA binding to silica. If the DNA solu-
tion increases the pH of the mixture, the color becomes
orange or violet. In this case the pH should be adjusted
1
Abbreviations used: PB, plasmid buer; GuHCl, guanidine
hydrochloride; MES, 2-(N-morpholine)ethanesulfonic acid; GuSCN,
guanidine thiocyanate, WB, wash buer; EB, elution buer; AB
absorption buer; NIB, NaI buer.

0003-2697/$ - see front matter 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0003-2697(03)00403-2
136 Notes & Tips / Analytical Biochemistry 321 (2003) 135137

(back to yellow) by a small amount (12 ll) of 3 M so-

dium acetate, pH 45.
The following buers are modications of published
recipies: PB4 and MgB [3], AB [4], and NIB [5].

Plasmid DNA (pDNA) purication

All purication methods listed in Tables 1 and 2 have

Fig. 1. (A) Preparation of homemade spin columns: original tube (1);
cutting of the cap and making the holes (2); sequential steps of the lter
insertion (35). (B) Elution of 5.5 kb plasmid from QIAprep (Qia) and
homemade columns (2 GF/F and 1 GF/F) by dierent volumes
of EB.
the same general steps. Nucleic acids are precipitated on
the column at slightly acidic pH in the presence of al-
cohol and chaotropic salt. Impurities are washed out by
chaotropic salt solution and, following this step, by 70
80% buered ethanol. The membrane is then dried by
brief centrifugation and pure DNA is eluted from the
column with elution buer. All centrifugation steps were
carried out in a tabletop centrifuge at maximum speed
(P10,000g) at normal temperature.
The protocol described in Table 1 is a modication of
the well-known alkaline lysis method. Buers PB1 and
PB2 are nearly the same as those in the classical proce-

Table 1
Protocol of plasmid DNA purication
1. Grow bacteria overnight in rich 2 YT medium with appropriate antibiotic.
2. Pellet 2 ml of bacterial culture by 2 min centrifugation and discard medium.
3. Resuspend pellet in 150 ll of PB1.
4. Add 150 ll of PB2 and mix thoroughly but gently.
5. Add 210 ll of PB3 and mix thoroughly for about 2 min.
6. Centrifuge for 510 min.
7. Load supernatant on the column and centrifuge 0.51.5 min.
8. Wash column by 400 ll of PB4 and centrifuge
0.5 min.
9. Wash twice by 400 ll of WB.
10. Discard eluate from 2-ml tube; dry out the column by 2 min centrifuging and place column in a clean 1.5-ml microfuge tube.
11. Add to the column
30 ll of EB and incubate P1 min.
12. Centrifuge for 2 min.

Table 2
Protocols for purication of DNA fragments
PCR purication
1. Add precipitation buer to PCR mixture and mix.
2. Load on the column.
3. Wash by 400 ll of PB4.
Purication of DNA from 1.5% agarose gels
1. Excise the DNA fragment from the gel.
2. Add four volumes of AB (or NIB) buer and incubate at 50 C for
10 min, occasionally vortexing.
3. After complete dissolving add one volume of isopropanol and mix.
4. Load on the column.
5. Wash column by 400 ll of AB (or NIB) (optional).
Separating double- (ds) and single-stranded (ss) DNA mixtures
1. Add ve volumes of PB4 to the DNA solution and mix.
2. Load the mixture on the column number 1 and collect eluate.
3. Wash column number 1 by 400 ll PB4.
4. Mix eluate from step 2 with half of the volume (relative to eluate) of MgB and load it on the column number 2.
As a result dsDNA is adsorbed on column number 1 and ssDNA is adsorbed on column number 2.
WB washing and DNA elution are the same as in Table 1.
 Notes & Tips / Analytical Biochemistry 321 (2003) 135137
dure [6]. The buer PB4 includes GuSCN. It is a more
active chaotropic agent than GuHCl, so PB4 washes out
some impurities that are resistant to PB3. EDTA was
included in the buers PB1, PB3, and PB4 to get rid of
RNA, irreversibly denaturated pDNA, and single-
stranded fragments of genomic DNA from the silica
membrane [3].
Purication of PCR products
For some applications the volume of precipitation
buer should be as small as possible. We have tried
several salt:isopropanol mixtures (Fig. 2B) in the pro-
tocol described in Table 2. It turned out, that one vol-
ume of NaI-based precipitation buer is sucient for
the purication of PCR fragments. This buer eciently
precipitates DNA fragments larger than 200 bp. Frag-
ments smaller than 100 bp are lost during loading or
PB4 washing (results not shown). For most applications,
the low yield of small DNA fragments is advantageous
because primers and primerdimers are removed.
Fig. 2. Application of the spin columns. In A, B, and C 1/20 part of the
reactions was loaded on gels. (A) Purication of 2.5-, 3.0-, 5.5-, and 12-
kbp plasmids on homemade columns with two GF/F membranes. The
lower part of the gel shows no RNA contaminations. (B) Purication
of 20 ll of PCR mix (
0.5 kb product in 50 mM TrisCl, pH 8.6,
50 mM KCl, 1.5 mM MgCl2 , 0.1% Tween 20, 1.5 M betaine) on 1
GF/F columns. Control without purication is marked by asterisk.
The volume of precipitation buer is shown above lanes. Precipitation
buers: 10 M LiCl:isopropanol [1:1] (LiCl); 5 M NaCl:isopropa-
nol [1:1] (NaCl); AB:isopropanol [1:1] (AB); NIB:isopropa-
nol [1:1] (NIB). (C) Separation of mixture (*) of double-stranded
pGEM4Z (lane 1) and single-stranded pBluescript II SK(+) (lane 2)
DNA. Eluted dsDNA is on lane 3; eluted ssDNA is on lane 4. (D)
Interference of silica purication with EcoRI digestion. About 0.2 lg
pDNA was digested by serial 2 dilutions of the restriction endonu-
clease EcoRI (30 min at 37 C; 1.25 U for lane 1). C, pDNA puried by
alkaline lysis method [11]; F, pDNA after additional GF/F purica-
tion; Q, pDNA after additional QIAprep purication.
137
Other applications
Both sodium iodide [5] and GuSCN are capable of
dissolving agarose at 50 C and may be used for puri-
cation of DNA fragments from agarose gels (Table 2).
The separation of single- and double-stranded DNA
mixture has been described previously for silica particles
[3]. Table 2 and Fig. 2C show the adaptation of this
procedure to spin columns.
Silica-based purication negatively aects some restric-
tion endonucleases
We have checked that the quality of DNA eluted from
homemade and Qiagen columns is similar for many ap-
plications, including sequencing, hybridization, labeling,
and PCR. However, in contrast to the generally accepted
opinion, we found that at least some restriction endo-
nucleases are sensitive to silica purication. Fig. 2D
demonstrates about a threefold inhibition of EcoRI en-
donuclease by a 1/30 volume of the eluted DNA. The in-
hibition varies depending on the endonuclease: HindII,
EcoRI, SphI, BglI, and NdeI are sensitive, but BamHI,
SacI, and NheI are completely insensitive. Centrifugation
of eluted DNA before aspiration can partly improve di-
gestion, suggesting that one reason for inhibition is tiny
glass dust stripped o from the membrane.
The suggested protocols for DNA purication are
cheap alternatives to commercial kits. They were suc-
cessfully used by our group and by our collaborators for
more than a year. The price for 250 columns and cor-
responding chemicals is about $35. It is therefore about
six times cheaper than available kits.
Acknowledgments
We thank Dr. I. Ivanov and Dr. H. Eickho for
helpful discussions. The work was supported by BMBF
Grant 0312275F/2.
References
[1] A.M. Prince, L. Andrus, PCR: how to kill unwanted DNA,
Biotechniques 12 (1992) 358360.
[2] B.H. Taylor, M.L. Nuccio, An internal indicator for alkaline
denaturation of double-stranded DNA in dideoxy sequencing
reactions, Biotechniques 14 (1993) 560.
[3] M. Beld, C. Sol, J. Goudsmit, R. Boom, Fractionation of nucleic
acids into single-stranded and double-stranded forms, Nucleic
Acids Res. 24 (1996) 26182619.
[4] R. Boom, C.J. Sol, M.M. Salimans, C.L. Jansen, P.M. Wertheim-
van Dillen, J. van der Noordaa, Rapid and simple method for
purication of nucleic acids, J. Clin. Microbiol. 28 (1990) 495503.
[5] M.L. Heyd, J.D. Diehl Jr., Recovering DNA from agarose gels
with pumice, Biotechniques 20 (1996) 394396, 398.
[6] S.Y. Lee, S. Rasheed, A simple procedure for maximum yield of
high-quality plasmid DNA, Biotechniques 9 (1990) 676679.