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Analytical Biochemistry 321 (2003) 135137
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We describe here the preparation and use of inex- Both homemade and recharged columns can sub-
pensive homemade spin columns which are at least as stitute for commercial columns in QIAprep and QIA-
ecient in purication of DNA as commercially avail- quick kits (results not shown). DNA binding capacity is
able kits. The minimal elution volume of homemade about 16 lg (8 lg) for 2(1) GF/F membranes. Because
columns is considerably smaller. The plasmid DNA of the compact geometry of the homemade columns
purication protocol proposed here does not involve the DNA may be eluted in a relatively small volume of
use of RNase, which is important for in vitro tran- elution buer (Fig. 1B).
scription experiments.
Buer solutions
Column preparation
All solutions are stable for at least 4 months at room
Silica membranes were cut out from GF/F borosili- temperature: plasmid buer (PB1),1 50 mM glucose,
cate glass ber paper (Whatman, England) by a 7-mm 35 mM EDTA, pH 8.0; PB2, 0.2 N NaOH, 1% SDS;
wad punch. GF/F has the largest DNA-binding capacity PB3 (should be prepared immediately before use from
(2.8 lg DNA/mg of membrane) compared to other types two stable components), four volumes [6.625 M guani-
of membranes: GF/A (0.10 lg/mg), GF/B (0.14 g/mg), dine hydrochloride (GuHCl), 62.5 mM 2-(N-morpho-
GF/C (0.46 lg/mg), and GF/D (0.13 lg/mg) (results not lino)ethanesulfonic acid (MES), pH in the interval 56,
shown). 0.3 M acetic acid, 25% (v/v) ethanol] and one volume
Homemade columns. Sequential steps of column [0.5 M EDTA, pH 8.0] (the nal mixture is stable for at
preparation are shown in Fig. 1A. The cap of a 0.5-ml least 1 day); PB4, 5 M guanidine thiocyanate (GuSCN),
microcentrifuge tube was cut o, but a small tail was 100 mM EDTA, pH 8.0; wash buer (WB), 10 mM Tris
left. Several holes were punctured in the bottom of the Cl, pH 7.5, 80% (v/v) ethanol; elution buer (EB),
tube by a red-hot needle. One or two membranes were 10 mM TrisCl, pH 8.5; adsorption buer (AB), 5 M
inserted tightly into the tube. For loading and washing GuSCN, 50 mM MES, pH 56, 20 mM EDTA, pH 8.0,
the column was placed in a reusable 2-ml screw-capped 0.5% Triton X-100, 0.05 mM cresol red; NaI buer
microcentrifuge tube; for DNA elution it was placed in a (NIB) 6.6 M NaI, 16 mM Na2 SO3 , 0.05 mM cresol red
1.5-ml tube. (store at 4 C in the dark) magnesium buer (MgB), 5 M
Recharging of used commercial columns (Qiagen). GuSCN, 100 mM MES, pH 56, 250 mM MgCl2 , 0.5%
The used membrane and plastic gasket ring were pushed Triton X-100.
out from the column by an unfolded paper clip. The Cresol red is included in AB and NIB buers as a pH
plastic parts were washed by detergent and water. To indicator dye [2]. It is yellow at pH 6 7.5, which is op-
prevent cross-contamination in sensitive applications, timal for the DNA binding to silica. If the DNA solu-
they can be treated with a 5% solution of sodium hy- tion increases the pH of the mixture, the color becomes
pochlorite [1]. Parts were dried; new membranes were orange or violet. In this case the pH should be adjusted
inserted and xed by the gasket ring.
1
Abbreviations used: PB, plasmid buer; GuHCl, guanidine
hydrochloride; MES, 2-(N-morpholine)ethanesulfonic acid; GuSCN,
*
Corresponding author. Fax: +49-30-8413-1128. guanidine thiocyanate, WB, wash buer; EB, elution buer; AB
E-mail address: soldatov@molgen.mpg.de (A.V. Soldatov). 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
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 137
dure [6]. The buer PB4 includes GuSCN. It is a more Other applications
active chaotropic agent than GuHCl, so PB4 washes out
some impurities that are resistant to PB3. EDTA was Both sodium iodide [5] and GuSCN are capable of
included in the buers PB1, PB3, and PB4 to get rid of dissolving agarose at 50 C and may be used for puri-
RNA, irreversibly denaturated pDNA, and single- cation of DNA fragments from agarose gels (Table 2).
stranded fragments of genomic DNA from the silica The separation of single- and double-stranded DNA
membrane [3]. mixture has been described previously for silica particles
[3]. Table 2 and Fig. 2C show the adaptation of this
procedure to spin columns.
Purication of PCR products
For some applications the volume of precipitation Silica-based purication negatively aects some restric-
buer should be as small as possible. We have tried tion endonucleases
several salt:isopropanol mixtures (Fig. 2B) in the pro-
tocol described in Table 2. It turned out, that one vol- We have checked that the quality of DNA eluted from
ume of NaI-based precipitation buer is sucient for homemade and Qiagen columns is similar for many ap-
the purication of PCR fragments. This buer eciently plications, including sequencing, hybridization, labeling,
precipitates DNA fragments larger than 200 bp. Frag- and PCR. However, in contrast to the generally accepted
ments smaller than 100 bp are lost during loading or opinion, we found that at least some restriction endo-
PB4 washing (results not shown). For most applications, nucleases are sensitive to silica purication. Fig. 2D
the low yield of small DNA fragments is advantageous demonstrates about a threefold inhibition of EcoRI en-
because primers and primerdimers are removed. 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