MinElute®
Handbook
MinElute PCR Purification Kit
For purification of PCR products (70 bp to 4 kb)
in low elution volumes
MinElute Gel Extraction Kit
For gel extraction of DNA fragments (70 bp to 4 kb)
in low elution volumes
MinElute Reaction Cleanup Kit
For cleanup of DNA (70 bp to 4 kb)
from enzymatic reactions
March 2008
Sample & Assay Technologies
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QIAGEN Sample and Assay Technologies
QIAGEN is the leading provider of innovative sample and assay technologies, enabling
the isolation and detection of contents of any biological sample. Our advanced,
high-quality products and services ensure success from sample to result.
QIAGEN sets standards in:
■ Purification of DNA, RNA, and proteins
■ Nucleic acid and protein assays
■ microRNA research and RNAi
■ Automation of sample and assay technologies
Our mission is to enable you to achieve outstanding success and breakthroughs. For
more information, visit www.qiagen.com .
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MinElute Handbook 03/2008 3
Contents
Kit Contents 4
Storage 5
Product Use Limitations 5
Product Warranty and Satisfaction Guarantee 5
Quality Control 5
Technical Assistance 6
Safety Information 7
Product Specifications 8
Introduction 9
The MinElute Principle 11
Equipment and Reagents to Be Supplied by User 18
MinElute PCR Purification Kit Protocols
using a microcentrifuge 19
using a vacuum manifold 21
MinElute Gel Extraction Kit Protocols
using a microcentrifuge 23
using a vacuum manifold 25
MinElute Reaction Cleanup Kit Protocols
using a microcentrifuge 28
using a vacuum manifold 30
Troubleshooting Guide 32
Appendix: QIAvac Vacuum Manifolds 34
References 36
Ordering Information 37
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4 MinElute Handbook 03/2008
Kit Contents
MinElute PCR Purification Kits (50) (250)
Catalog no. 28004 28006
MinElute Spin Columns 50 250
Buffer PB* 30 ml 150 ml
Buffer PE (concentrate) 2 x 6 ml 55 ml
Buffer EB 15 ml 15 ml
pH Indicator 800 µl 800 µl
Collection Tubes (2 ml) 50 250
Loading Dye 110 µl 550 µl
Handbook 1 1
MinElute Gel Extraction Kits (50) (250)
Catalog no. 28604 28606
MinElute Spin Columns 50 250
Buffer QG* 2 x 50 ml 2 x 250 ml
Buffer PE (concentrate) 2 x 6 ml 55 ml
Buffer EB 15 ml 15 ml
Collection Tubes (2 ml) 50 250
Loading Dye 110 µl 550 µl
Handbook 1 1
MinElute Reaction Cleanup Kits (50) (250)
Catalog no. 28204 28206
MinElute Spin Columns 50 250
Buffer ERC* 20 ml 85 ml
Buffer PE 2 x 6 ml 55 ml
Buffer EB 15 ml 15 ml
Collection Tubes (2 ml) 50 250
Loading Dye 110 µl 550 µl
Handbook 1 1
* Buffers PB, QG, and ERC contain chaotropic salts which are irritants. Take appropriate laboratory safety
measures and wear gloves when handling.
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MinElute Handbook 03/2008 5
Storage
Upon arrival, open the kit and store MinElute spin columns at 2–8°C. The remaining kit
components can be stored at room temperature (15–25°C). Under these conditions,
MinElute Kits can be stored for up to 12 months without showing any reduction in
performance and quality. Check buffers for precipitate before use and redissolve at 37°C
if necessary. The entire kit can be stored at 2–8°C, but in this case buffers should be
redissolved before use. Make sure that all buffers are at room temperature when used.
Product Use Limitations
MinElute Kits are developed, designed and sold for research purposes only. They are not
to be used for human diagnostic or drug purposes or to be administered to humans unless
expressly cleared for that purpose by the Food and Drug Administration in the USA or the
appropriate regulatory authorities in the country of use. All due care and attention should
be exercised in the handling of many of the materials described in this text.
Product Warranty and Satisfaction Guarantee
QIAGEN guarantees the performance of all products in the manner described in our
product literature. The purchaser must determine the suitability of the product for its
particular use. Should any product fail to perform satisfactorily due to any reason other than
misuse, QIAGEN will replace it free of charge or refund the purchase price. We reserve
the right to change, alter, or modify any product to enhance its performance and design.
If a QIAGEN®
product does not meet your expectations, simply call your local Technical
Service Department. We will credit your account or exchange the product — as you wish.
Separate conditions apply to QIAGEN scientific instruments, service products, and to
products shipped on dry ice. Please inquire for more information.
A copy of QIAGEN terms and conditions can be obtained on request, and is also
provided on the back of our invoices. If you have questions about product specifications or
performance, please call QIAGEN Technical Services or your local distributor (see back
cover or visit www.qiagen.com ).
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of
MinElute Kits is tested against predetermined specifications to ensure consistent
product quality.
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6 MinElute Handbook 03/2008
Technical Assistance
At QIAGEN we pride ourselves on the quality and availability of our technical support. Our
Technical Service Departments are staffed by experienced scientists with extensive
practical and theoretical expertise in molecular biology and the use of QIAGEN products.
If you have any questions or experience any problems regarding any aspect of MinElute
Kits, or QIAGEN products in general, please do not hesitate to contact us.
QIAGEN customers are a major source of information regarding advanced or specialized
uses of our products. This information is helpful to other scientists as well as to the
researchers at QIAGEN. We therefore encourage you to contact us if you have any
suggestions about product performance or new applications and techniques.
For technical assistance and more information please call one of the QIAGEN
Technical Service Departments or local distributors (see back cover or visit
www.qiagen.com ).
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MinElute Handbook 03/2008 7
Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves,
and protective goggles. For more information, please consult the appropriate material
safety data sheets (MSDSs). These are available online in convenient and compact PDF
format at www.qiagen.com/ts/msds.asp where you can find, view, and print the MSDS
for each QIAGEN kit and kit component.
CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation
waste.
Buffers PB and ERC contain guanidine hydrochloride, which can form highly reactive
compounds when combined with bleach.
In case liquid containing these buffers is spilt, clean with suitable laboratory detergent
and water. If the spilt liquid contains potentially infectious agents, clean the affected area
first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.
The following risk and safety phrases apply to the components of the MinElute DNA
Cleanup System
Buffers PB
Contains guanidine hydrochloride and isopropanol: harmful, irritant, flammable. Risk and
safety phrases:* R10-22-36/38, S23-26-36/37/39-46
Buffer ERC
Contains guanidine hydrochloride and isopropanol: harmful, irritant, flammable. Risk and
safety phrases:* R10-22-36/38, S23-26-36/37/39-46
Buffer QG
Contains guanidine thiocyanate: harmful, irritant. Risk and safety phrases:* R20/21/
22-32, S13-26-36-46
24-hour emergency information
Emergency medical information in English, French, and German can be obtained
24 hours a day from:
Poison Information Center Mainz, Germany
Tel: +49-6131-19240
* R10: Flammable. R22: Harmful if swallowed. R20/21/22: Harmful by inhalation, in contact with skin and
if swallowed. R32: Contact with acids liberates very toxic gas. R36/38: Irritating to eyes and skin. S13:
Keep away from food, drink and animal feedingstuffs. S23: Do not breathe vapour/spray. S26: In case of
contact with eyes, rinse immediately with plenty of water and seek medical advice. S36: Wear suitable
protective clothing. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S46:
If swallowed, seek medical advice immediately and show the container or label.
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8 MinElute Handbook 03/2008
MinElute MinElute MinElute
PCR Purification Kit Gel Extraction Kit Reaction Cleanup Kit
Maximum binding
capacity: 5 µg 5 µg 5 µg
Recovery of DNA: 80% 80% 80%
(70 bp – 4kb) (70 bp – 4 kb) (70 bp – 4 kb)
Maximum weight
of gel slice: — 400 mg —
Elution volume: 10 µl 10 µl 10 µl
Volume of eluate: 9 µl 9 µl 9 µl
Capacity of column
reservoir: 800 µl 800 µl 800 µl
Recovered:
dsDNA 70 bp – 4 kb 70 bp – 4 kb 70 bp – 4 kb
Removed:
<40mers YES n.a. YES
Product Specifications
n.a.: not applicable
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MinElute Handbook 03/2008 9
Table 1. Enzymes Commonly Used in Enzymatic Manipulation of DNA
Protein Molecular weight per enzyme subunit (kDa)
DNA Polymerase I 109
Klenow fragment 62
Calf intestinal alkaline phosphatase (CIP) 69
T4 DNA ligase 55
T4 Polynucleotide kinase 35
Terminal transferase 32
DNase I 31
Introduction
The MinElute DNA Cleanup system is specially designed for fast and easy isolation of DNA
fragments from PCR reactions, agarose gels, or enzymatic reactions with an extremely small
elution volume of only 10 µl. Designed for rapid DNA cleanup, with extremely high
end-concentration of DNA, the MinElute system includes:
■ MinElute PCR Purification Kits for direct purification of double-stranded PCR
products (70 bp – 4 kb) from amplification reactions.
■ MinElute Gel Extraction Kits for extraction of DNA fragments (70 bp – 4 kb)
from standard or low-melt agarose gels in TAE (Tris-acetate/EDTA) or TBE
(Tris-borate/EDTA) buffer.
■ MinElute Reaction Cleanup Kits to purify double-stranded DNA fragments
(70 bp – 4 kb) from all enzymatic reactions.
The MinElute Reaction Cleanup Kit can be used for DNA cleanup from all enzymatic
reactions, including:
■ Dephosphorylation ■ Primed synthesis
■ Restriction enzyme digestion ■ Endlabeling
■ Ligation ■ Nick translation
During DNA cleanup using MinElute Reaction Cleanup Kits, all enzymes are removed,
independent of size and secondary structure. Table 1 lists examples of enzymes that
are used in the above reactions and are removed, together with salts and oligomers,
during the MinElute Reaction Cleanup procedure.
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10 MinElute Handbook 03/2008
MinElute Kits provide high yields of pure nucleic acids in minimal elution volumes. The
highly concentrated DNA is suitable for direct use in applications such as:
■ Ligation and transformation ■ In vitro transcription
■ Radioactive and fluorescent sequencing ■ Amplification
■ Restriction enzyme digestion ■ Microinjection
■ Labeling ■ Hybridization
Figure. DNA fragment binding-size range. Recoveries of DNA fragments in the size range between “removed”
and “recovered” are not defined.
Automated DNA cleanup
The MinElute PCR Purification Kit and MinElute Gel Extraction Kit can be fully
automated on the QIAcube. The innovative QIAcube uses advanced technology to
process QIAGEN spin columns, enabling seamless integration of automated,
low-throughput sample prep into your laboratory workflow. Sample preparation using
the QIAcube follows the same steps as the manual procedure (i.e., bind, wash, and
elute) enabling purification of high-quality DNA. The QIAcube is preinstalled with
protocols for purification of plasmid DNA, genomic DNA, RNA, viral nucleic acids, and
proteins, plus DNA and RNA cleanup. The range of protocols available is continually
expanding, and additional QIAGEN protocols can be downloaded free of charge at
www.qiagen.com/MyQIAcube . A detailed protocol for using QIAquick spin columns
on the QIAcube is provided with the QIAcube.
Note: It is not necessary to add pH indicator I to Buffer PB when using the QIAcube.
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MinElute Handbook 03/2008 11
The MinElute Principle
The MinElute system combines the convenience of spin-column technology with the
selective binding properties of a uniquely designed silica membrane. MinElute
columns are designed to give high end-concentrations of purified DNA fragments for
subsequent reactions. Special buffers provided with each kit are optimized for efficient
recovery of DNA and removal of contaminants in each specific application. DNA
adsorbs to the silica membrane in the presence of high concentrations of salt while
contaminants pass through the column. Impurities are efficiently washed away, and the
pure DNA is eluted with Tris buffer or water (see page 17). MinElute columns offer
two handling options — as an alternative to processing the spin columns in a
microcentrifuge, they can also be used on any commercial vacuum manifold with luer
connectors (e.g., QIAvac 24 Plus or QIAvac 6S with QIAvac Luer Adapters). The
uniquely designed MinElute column with specialized membrane assembly allows
purification of concentrated DNA fragments in high yields, in as little as 9 µl eluate
(Figure 1).
Unique MinElute
membrane assembly
Figure 2. Unique membrane assembly enables low elution volumes. Cross-section of MinElute column.
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12 MinElute Handbook 03/2008
Adsorption to MinElute membrane — salt and pH dependence
The MinElute silica membrane is uniquely adapted to purify DNA from both
aqueous solutions and agarose gels, and up to 5 µg DNA can bind to each MinElute
column. The binding buffers in MinElute Spin Kits provide the correct salt concentration
and pH for adsorption of DNA to the MinElute membrane. The adsorption of nucleic acids
to silica surfaces occurs only in the presence of a high concentration of chaotropic salts
(1), which modify the structure of water (2). Adsorption of DNA to silica also depends on
pH. Adsorption is typically 80% if the pH is ≤7.5, and is reduced drastically at higher pH
(Figure 2). If the loading mixture pH is >7.5, the optimal pH for DNA binding can be
obtained by adding a small volume of 3 M sodium acetate, pH 5.0.
Figure 3. pH dependence of DNA adsorption to MinElute membranes pH dependence of DNA adsorption
to silica. 1 µg of a 2.9 kb DNA fragment was adsorbed at different pHs and eluted with Buffer EB (10 mM
Tris·Cl, pH 8.5). The graph shows the percentage of DNA recovery, reflecting the relative adsorption
efficiency, versus pH of adsorption.
Optimized binding buffers for every DNA cleanup task
MinElute Kits contain identical MinElute columns and different binding buffers optimized
for each specific application:
■ Buffer PB in the MinElute PCR Purification Kit allows the efficient binding of
double-stranded PCR products as small as 70 bp and the removal of primers up
to 40 nucleotides.
■ Buffer QG in the MinElute Gel Extraction Kit solubilizes the agarose gel slice and
provides the appropriate conditions for binding of DNA to the silica membrane.
■ Buffer ERC in the MinElute Reaction Cleanup Kit allows the efficient binding of double
stranded DNA as small as 70 bp and the removal of enzymes, salts, and oligomers.
■ Buffer PB and Buffer PBI and Buffer QG are also available separately (see Ordering
Information).
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MinElute Handbook 03/2008 13
pH indicator
Buffers PB, QG, and ERC are specially optimized for use with the MinElute silica
membrane. Buffers QG and ERC contain an integrated pH indicator, while an optional
pH indicator can be added to Buffer PB allowing easy determination of the optimal pH
for DNA binding. DNA adsorption requires a pH Յ7.5, and the pH indicator in the
buffers will appear yellow in this range. If the pH is >7.5, which can occur if during
agarose gel electrophoresis, the electrophoresis buffer had been used repeatedly or
incorrectly prepared, or if the buffer used in an enzymatic reaction is strongly basic
and has a high buffering capacity, the binding mixture turns orange or violet (Figure
2). This means that the pH of the sample exceeds the buffering capacity of Buffer PB,
QG, or ERC and DNA adsorption will be inefficient. In these cases, the pH of the
binding mixture can easily be corrected by addition of a small volume of 3 M sodium
acetate*, pH 5.0, before proceeding with the protocol. In addition, in the MinElute
Gel Extraction Kit procedure, the color of the binding mixture allows easy visualization
of any unsolubilized agarose, ensuring complete solubilization and maximum yields.
The indicator dye does not interfere with DNA binding and is completely removed
during the cleanup procedure. Buffers PB, QG, and ERC do not contain sodium iodide
(NaI). Residual NaI may be difficult to remove from DNA samples, and reduces the
efficiency of subsequent enzymatic reactions such as blunt-end ligation.
Optimal pH pH too high
Figure 4. Indicator enables easy checking of the optimal pH. Indicator dye in solubilization and binding Buffers
PBI, QG, and ERC identifies optimal pH for DNA binding.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, please consult the appropriate material safety data sheets (MSDSs) available from the
product supplier.
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14 MinElute Handbook 03/2008
Washing
During the DNA adsorption step, unwanted primers and impurities, such as salts,
enzymes, unincorporated nucleotides, agarose, dyes, ethidium bromide, DMSO, oils,
and detergents (e.g., Tween®
20) do not bind to the silica membrane, but flow through
the column. Salts are quantitatively washed away by the ethanol-containing Buffer PE.
Any residual Buffer PE, which may interfere with subsequent enzymatic reactions, is
removed by an additional centrifugation step.
Elution in low-salt solutions
Elution efficiency is strongly dependent on the salt concentration and pH of the elution
buffer. Contrary to adsorption, elution is most efficient under basic conditions and low
salt concentrations. DNA is eluted with 10 µl of the provided Buffer EB (10 mM Tris·Cl,
pH 8.5) or water. The volume of the final eluate is 9 µl. The maximum elution efficiency
is achieved between pH 7.0 and 8.5. When using water to elute, make sure that the
pH is within this range. In addition, DNA must be stored at –20°C when eluted with
water since DNA may degrade in the absence of a buffering agent. Elution with TE
buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0) is possible, but not recommended because
EDTA may inhibit subsequent enzymatic reactions.
Agarose gel analysis of yield
Yields of DNA following cleanup can be analyzed by agarose gel analysis. Table 2
shows the total yield obtained following extraction of 1 µg or 0.5 µg starting DNA from
an agarose gel with a recovery of 80% using the MinElute Gel Extraction Kit. The
corresponding amount of DNA in a 1 µl aliquot from 10 µl elution volume (9 µl eluate)
is indicated.
Table 2. Amount of DNA in 1 µl Aliquots of a 9 µl Eluate Following MinElute Purification
Amount of
Total yield DNA in 1 µl
Starting DNA Recovery (10 µl elution volume) (9 µl eluate)
1 µg 80% 800 ng 89 ng
0.5 µg 80% 400 ng 44 ng
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MinElute Handbook 03/2008 15
Quantification of DNA fragments
DNA fragments can be quantified by running a sample alongside standards containing
known quantities of the same-sized DNA fragment. The amount of sample DNA loaded can
be estimated by visual comparison of the band intensity with that of the standards
(Figure 4).
Figure 5. Agarose gel analysis. An unknown amount of a 5.5 kb DNA fragment (U) was run alongside known
quantities (as indicated in ng) of the same DNA fragment. The unknown sample contained 75–100 ng DNA,
as estimated by visual comparison with the standards. M: 1 kb DNA ladder.
Loading dye
Loading dye is provided for analysis of purified DNA samples using electrophoresis.
It contains 3 marker dyes (bromophenol blue, xylene cyanol, and orange G) that
facilitate estimation of DNA migration distance and optimization of agarose gel run
time. Refer to Table 3 to identify the dyes according to migration distance and agarose
gel percentage and type. Loading dye is supplied as a 5x concentrate; thus 1 volume
of loading dye should be added to 5 volumes of purified DNA.
M 125 ng 100 ng 75 ng 50 ng U
Table 3. Migration Distance of Gel Tracking Dyes
%TAE (TBE) Xylene cyanol Bromophenol blue Orange G
agarose gel (light blue) (dark blue) (orange)
0.8 5000 bp (3000 bp) 800 bp (400 bp) 150 bp (<100 bp)
1.0 3000 bp (2000 bp) 400 bp (250 bp) <100 bp (<100 bp)
1.5 1800 bp (1100 bp) 250 bp (100 bp) <100 bp (<100 bp)
2.0 1000 bp (600 bp) 200 bp (<100 bp) <100 bp (<100 bp)
2.5 700 bp (400 bp) 100 bp (<50 bp) <50 bp (<50 bp)
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16 MinElute Handbook 03/2008
Applications using MinElute purified DNA
DNA purified by the MinElute system is significantly more concentrated than DNA
purified by other methods. The highly concentrated DNA makes small reaction volumes
possible for downstream applications, leading to increased efficiency (e.g., in
ligations). DNA purified with MinElute Kits is suitable for any subsequent application,
such as ligation and transformation, radioactive and fluorescent sequencing, restriction
enzyme digestion, labeling, hybridization, PCR, in vitro transcription, or microinjection.
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MinElute Handbook 03/2008 17
PCR or other
enzymatic reaction or
solubilized gel slice
Pure DNA fragment
Vacuum
Vacuum
QIAcube
Optimal pH pH too high
The MinElute Procedure
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18 MinElute Handbook 03/2008
Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves,
and protective goggles. For more information, please consult the appropriate material
safety data sheets (MSDSs) available from the product supplier.
For all protocols
■ Ethanol (96–100%)*
■ Microcentrifuge
■ 1.5 or 2 ml microcentrifuge tubes
■ 3 M sodium acetate, pH 5.0, may be necessary for PCR purification and gel
extraction protocols.
■ Optional: Distilled water or TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH8) for
elution of DNA.
Vacuum protocols
■ Vacuum manifold (e.g., QIAvac 24 Plus or QIAvac 6S)
■ Vacuum pump (e.g., QIAGEN Vacuum Pump, see ordering information).
Gel extraction protocols
■ Isopropanol (100%)
■ Heating block or water bath set at 50°C
* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.
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MinElute Handbook 03/2008 19
MinElute PCR Purification Kit Protocol
using a microcentrifuge
This protocol is designed to purify double-stranded DNA fragments from PCR reactions
resulting in high end-concentrations of DNA (see page 12). Fragments ranging from
70 bp to 4 kb are purified from primers, nucleotides, polymerases, and salts using
MinElute spin columns in a microcentrifuge.
Important points before starting
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a
conventional tabletop microcentrifuge at room temperature.
■ Add 1:250 volume pH indicator I to Buffer PB (i.e., add 120 µl pH indicator I to
30 ml Buffer PB or add 600 µl pH indicator I to 150 ml Buffer PB). The yellow
color of Buffer PB with pH indicator I indicates a pH of Յ7.5.
■ Add pH indicator I to entire buffer contents. Do not add pH indicator I to buffer
aliquots.
■ If the purified PCR product is to be used in sensitive microarray applications, it
may be beneficial to use Buffer PB without the addition of pH indicator I.
Procedure
1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. It is not
necessary to remove mineral oil or kerosene.
For example, add 250 µl of Buffer PB to 50 µl PCR reaction (not including oil).
2. If pH indicator I has been added to Buffer PB, check that the color of the mixture
is yellow.
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate,
pH 5.0, and mix. The color of the mixture will turn to yellow.
3. Place a MinElute column in a provided 2 ml collection tube in a suitable rack.
4. To bind DNA, apply the sample to the MinElute column and centrifuge for 1 min.
For maximum recovery, transfer all traces of sample to the column.
5. Discard flow-through. Place the MinElute column back into the same tube.
6. To wash, add 750 µl Buffer PE to the MinElute column and centrifuge for 1 min.
7. Discard flow-through and place the MinElute column back in the same tube.
Centrifuge the column for an additional 1 min at maximum speed.
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed
unless the flow-through is discarded before this additional centrifugation.
8. Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
PCRPurification
SpinProtocol
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20 MinElute Handbook 03/2008
9. To elute DNA, add 10 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center
of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the center of
the membrane for complete elution of bound DNA. The average eluate volume is
9 µl from 10 µl elution buffer volume.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. When using water, make sure that the pH value is within
this range, and store DNA at –20°C as DNA may degrade in the absence of a
buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl,
1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to
5 volumes of purified DNA. Mix the solution by pipetting up and down before
loading the gel.
Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and
orange G) that facilitate estimation of DNA migration distance and optimization
of agarose gel run time. Refer to Table 3 (page 15) to identify the dyes according
to migration distance and agarose gel percentage and type.
PCRPurification
SpinProtocol
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PCRPurification
VacuumProtocol
MinElute Handbook 03/2008 21
MinElute PCR Purification Kit Protocol
using a vacuum manifold
MinElute spin columns can also be used on any vacuum manifold with luer connectors (e.g.,
QIAvac 24 Plus or QIAvac 6S with Luer Adapters). The following protocol is designed to
purify double-stranded DNA fragments from PCR reactions resulting in high endconcentrations
of DNA (see page 12). Fragments ranging from 70 bp to 4 kb are purified
from primers, nucleotides, polymerases and salts using vacuum-driven sample processing.
Important points before starting
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ Switch off vacuum between steps to ensure that a consistent, even vacuum is
applied during manipulations.
■ Add 1:250 volume pH indicator I to Buffer PB (i.e., add 120 µl pH indicator I to
30 ml Buffer PB or add 600 µl pH indicator I to 150 ml Buffer PB). The yellow color
of Buffer PB with pH indicator I indicates a pH of Յ7.5.
■ Add pH indicator I to entire buffer contents. Do not add pH indicator I to buffer
aliquots.
■ If the purified PCR product is to be used in sensitive microarray applications, it may
be beneficial to use Buffer PB without the addition of pH indicator I.
Procedure
1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. It is not
necessary to remove mineral oil or kerosene.
For example, add 250 µl of Buffer PB to 50 µl PCR reaction (not including oil).
2. If pH indicator 1 has been added to Buffer PB, check that the color of the mixture
is yellow.
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate,
pH 5.0, and mix. The color of the mixture will turn to yellow.
3. Prepare the vacuum manifold and MinElute columns according to step 3a, 3b,
or 3c.
3a. QIAvac 24 Plus (see page 34):
Insert up to 24 MinElute spin columns into the luer extensions of the QIAvac 24
Plus. Close unused positions with luer caps and connect QIAvac 24 Plus to a
vacuum source.
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22 MinElute Handbook 03/2008
3b. QIAvac 6S manifold (see page 35):
Open QIAvac 6S lid. Place QIAvac Luer Adapter(s), or blanks to seal unused slots,
into the slots of QIAvac top plate, and close the QIAvac 6S lid. Place the waste
tray inside the QIAvac base, and place the top plate squarely over the base.
Attach the QIAvac 6S to a vacuum source.
Insert each MinElute column into a luer connector on the Luer Adapter(s) in the
manifold. Seal unused luer connectors with plugs provided with the QIAvac Luer
Adapter Set.
3c. Other vacuum manifolds: follow the supplier's instructions. Insert each MinElute
column into a luer connector.
4. To bind DNA, load the samples into the MinElute columns by decanting or pipetting,
and apply vacuum. After the samples have passed through the columns, switch off
the vacuum source.
For maximum recovery, transfer all traces of sample to the column.
The maximum loading volume of the column is 800 µl. For sample volumes greater
than 800 µl simply load again.
5. To wash, add 750 µl of Buffer PE to each MinElute column and apply vacuum.
6. Transfer each MinElute column to a microcentrifuge tube or the provided 2 ml
collection tubes. Centrifuge for 1 min at ≥10,000 x g.
IMPORTANT: This spin is necessary to remove residual ethanol (Buffer PE).
7. Place each MinElute column into a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 10 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center
of each membrane, let the columns stand for 1 min, and then centrifuge.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the center of
the membrane for complete elution of bound DNA. The average eluate volume is
9 µl from 10 µl elution buffer volume.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. When using water, make sure that the pH value is within
this range, and store DNA at –20°C as DNA may degrade in the absence of a
buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl,
1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
9. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to
5 volumes of purified DNA. Mix the solution by pipetting up and down before
loading the gel.
Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and
orange G) that facilitate estimation of DNA migration distance and optimization
of agarose gel run time. Refer to Table 3 (page 15) to identify the dyes according
to migration distance and agarose gel percentage and type.
PCRPurification
VacuumProtocol
1051747_HB 15.04.2008 13:24 Uhr Seite 22
GelExtraction
SpinProtocol
MinElute Handbook 03/2008 23
MinElute Gel Extraction Kit Protocol
using a microcentrifuge
This protocol is designed to extract and purify DNA of 70 bp to 4 kb from standard or
low-melt agarose gels in TAE or TBE buffer resulting in high end-concentrations of DNA.
Up to 400 mg agarose can be processed per MinElute column.
Important points before starting
■ The yellow color of Buffer QG indicates a pH ≤7.5.
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ All centrifugation steps are carried out at ≥10,000 x g in a conventional table-top
microcentrifuge at room temperature.
Procedure
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of
gel (100 mg or approximately 100 µl).
For example, add 300 µl of Buffer QG to each 100 mg of gel. For >2% agarose
gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per spin
column is 400 mg; for gel slices >400 mg use more than one MinElute column.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help
dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is
yellow (similar to Buffer QG without dissolved agarose).
Note: If the color of the mixture is orange or violet, add 10 µl of 3 M sodium
acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
The adsorption of DNA to the membrane is efficient only at pH ≤7.5. Buffer QG
contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher
pH, allowing easy determination of the optimal pH for DNA binding.
5. Add 1 gel volume of isopropanol to the sample and mix by inverting the tube
several times.
For example, if the agarose gel slice is 100 mg, add 100 µl isopropanol. Do not
centrifuge the sample at this stage.
6. Place a MinElute column in a provided 2 ml collection tube in a suitable rack.
1051747_HB 15.04.2008 13:24 Uhr Seite 23
24 MinElute Handbook 03/2008
GelExtraction
SpinProtocol
7. To bind DNA, apply the sample to the MinElute column, and centrifuge for 1 min.
For maximum recovery, transfer all traces of sample to the column. The maximum
volume of the column reservoir is 800 µl. For sample volumes of more than
800 µl, simply load and spin again.
8. Discard the flow-through and place the MinElute column back in the same collection
tube.
9. Add 500 µl of Buffer QG to the spin column and centrifuge for 1 min.
10. Discard the flow-through and place the MinElute column back in the same collection
tube.
11. To wash, add 750 µl of Buffer PE to the MinElute column and centrifuge for 1 min.
Note: If the DNA will be used for salt-sensitive applications, such as blunt-end
ligation and direct sequencing, let the column stand 2–5 min after addition of
Buffer PE, before centrifuging.
12. Discard the flow-through and centrifuge the MinElute column for an additional
1 min at ≥10,000 x g.
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed
unless the flow-through is discarded before this additional centrifugation.
13. Place the MinElute column into a clean 1.5 ml microcentrifuge tube.
14. To elute DNA, add 10 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center
of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the center of
the membrane for complete elution of bound DNA. The average eluate volume is
9 µl from 10 µl elution buffer volume.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. When using water, make sure that the pH value is within
this range, and store DNA at –20°C as DNA may degrade in the absence of a
buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl,
1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
15. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to
5 volumes of purified DNA. Mix the solution by pipetting up and down before
loading the gel.
Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and
orange G) that facilitate estimation of DNA migration distance and optimization
of agarose gel run time. Refer to Table 3 (page 15) to identify the dyes according
to migration distance and agarose gel percentage and type.
1051747_HB 15.04.2008 13:24 Uhr Seite 24
GelExtraction
VacuumProtocol
MinElute Handbook 03/2008 25
MinElute Gel Extraction Kit Protocol
using a vacuum manifold
MinElute columns can also be used on any vacuum manifold with luer connectors (e.g.,
QIAvac 24 Plus or QIAvac 6S with Luer Adapters). The following protocol is designed
to extract and purify DNA of 70 bp to 4 kb from standard or low-melt agarose gels in
TAE or TBE buffer using vacuum-driven processing resulting in high end-concentrations
of DNA. Up to 400 mg agarose can be processed per MinElute column.
Important points before starting
■ The yellow color of Buffer QG indicates a pH ≤7.5.
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ Switch off vacuum between steps to ensure that a consistent, even vacuum is
applied during manipulations.
Procedure
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of
gel (100 mg or approximately 100 µl).
For example, add 300 µl of Buffer QG to each 100 mg of gel. For >2% agarose
gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per spin
column is 400 mg; for gel slices >400 mg use more than one spin column.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help
dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
4. During the incubation, prepare the vacuum manifold and MinElute columns
according to steps 4a, 4b, or 4c.
4a. QIAvac 24 Plus (see page 34):
Insert up to 24 MinElute columns into the luer extensions of the QIAvac 24 Plus.
Close unused positions with luer caps and connect QIAvac 24 Plus to a vacuum
source.
1051747_HB 15.04.2008 13:24 Uhr Seite 25
GelExtraction
VacuumProtocol
26 MinElute Handbook 03/2008
4b. QIAvac 6S manifold (see page 35):
Open QIAvac 6S lid. Place QIAvac Luer Adapter(s), or blanks to seal unused slots,
into the slots of QIAvac top plate, and close the QIAvac 6S lid. Place the waste
tray inside the QIAvac base, and place the top plate squarely over the base.
Attach the QIAvac 6S to a vacuum source.
Insert each MinElute column into a luer connector on the Luer Adapter(s) in the
manifold. Seal unused luer connectors with plugs provided with the QIAvac Luer
Adapter Set.
4c. Other vacuum manifolds: follow the suppliers instructions. Insert each MinElute
column into a luer connector.
5. After the gel slice has dissolved completely, check that the color of mixture is yellow
(similar to Buffer QG without dissolved agarose).
Note: If the color of the sample is orange or violet, add 10 µl of 3 M sodium
acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
The adsorption of DNA to the membrane is efficient only at pH ≤7.5.
Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet
at higher pH, allowing easy determination of the optimal pH for DNA binding.
6. Add 1 gel volume of isopropanol to the sample and mix by inverting the tube
several times.
For example, if the agarose gel slice is 100 mg, add 100 µl isopropanol. Do not
centrifuge the sample at this stage.
7. To bind DNA, pipet the sample onto the MinElute column and apply vacuum. After
the sample has passed through the column, switch off vacuum source.
For maximum recovery, transfer all traces of sample to the column.
The maximum volume of the column reservoir is 800 µl. For sample volumes of
more than 800 µl, simply load again.
8. Add 500 µl of Buffer QG to the MinElute column and apply vacuum.
9. To wash, add 750 µl of Buffer PE to the MinElute column and apply vacuum.
Note: If the DNA will be used for salt-sensitive applications, such as blunt-end
ligation and direct sequencing, let the column stand 2–5 min after addition of
Buffer PE before applying vacuum.
10. Transfer the MinElute column to a clean 1.5 ml microfuge tube or to a provided
2 ml collection tube. Centrifuge for 1 min at ≥≥10,000 x g.
IMPORTANT: This spin is necessary to remove residual ethanol (Buffer PE).
11. Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
1051747_HB 15.04.2008 13:24 Uhr Seite 26
MinElute Handbook 03/2008 27
12. To elute DNA, add 10 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center
of the membrane, let the columns stand for 1 min, and then centrifuge for
1 min.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the center of
the membrane for complete elution of bound DNA. The average eluate volume is
9 µl from 10 µl elution buffer volume.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. When using water, make sure that the pH value is within
this range, and store DNA at –20°C as DNA may degrade in the absence of a
buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl,
1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
13. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to
5 volumes of purified DNA. Mix the solution by pipetting up and down before
loading the gel.
Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and
orange G) that facilitate estimation of DNA migration distance and optimization
of agarose gel run time. Refer to Table 3 (page 15) to identify the dyes according
to migration distance and agarose gel percentage and type.
GelExtraction
VacuumProtocol
1051747_HB 15.04.2008 13:24 Uhr Seite 27
ReactionCleanup
SpinProtocol
28 MinElute Handbook 03/2008
MinElute Reaction Cleanup Kit Protocol
using a microcentrifuge
This protocol is designed to purify double-stranded DNA fragments from all enzymatic
reactions e.g., restriction digestion and labeling resulting in high end-concentrations
of DNA (see page 12). Fragments ranging from 70 bp to 4 kb are purified from
enzymes, primers, nucleotides, and salts using the MinElute Reaction Cleanup columns
in a microcentrifuge. The DNA-binding capacity of the MinElute columns is 5 µg.
Important points before starting
■ The yellow color of Buffer ERC indicates a pH ≤7.5.
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ All centrifuge steps are carried out at ≥10,000 x g in a conventional tabletop
microcentrifuge at room temperature.
Procedure
1. Add 300 µl of Buffer ERC to the enzymatic reaction and mix. The maximum
volume of enzymatic reaction that can be processed per MinElute column is 100 µl.
If the enzymatic reaction is in a volume of <20 µl, adjust the volume to 20 µl. If
the enzymatic reaction exceeds 100 µl, split your reaction, add 300 µl of Buffer
ERC to each aliquot of the split reaction, and use the appropriate number of
MinElute columns.
2. Check that the color of the mixture is yellow (similar to Buffer ERC without the
enzymatic reaction).
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate,
pH 5.0, and mix. The color of the mixture will turn to yellow.
3. Place a MinElute column in a 2 ml collection tube in a suitable rack.
4. To bind DNA, apply the sample to the MinElute column and centrifuge for 1 min.
To obtain maximal recovery, transfer all traces of sample to the spin column.
5. Discard the flow-through and place the MinElute column back into the same tube.
6. To wash, add 750 µl Buffer PE to the MinElute column and centrifuge for 1 min.
7. Discard the flow-through and place the MinElute column back in the same tube.
Centrifuge the column for an additional 1 min at maximum speed.
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed
unless the flow-through is discarded before this additional centrifugation.
8. Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
1051747_HB 15.04.2008 13:24 Uhr Seite 28
MinElute Handbook 03/2008 29
9. To elute DNA, add 10 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center
of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the
membrane for complete elution of bound DNA. The average volume of eluate is
9 µl from 10 µl elution buffer. Elution efficiency is dependent on pH. The maximum
elution efficiency is achieved between pH 7.0 and 8.5. When using water, make
sure that the pH value is within this range, and store DNA at –20°C as DNA may
degrade in the absence of a buffering agent. The purified DNA can also be eluted
in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent
enzymatic reactions.
10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to
5 volumes of purified DNA. Mix the solution by pipetting up and down before
loading the gel.
Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and
orange G) that facilitate estimation of DNA migration distance and optimization
of agarose gel run time. Refer to Table 3 (page 15) to identify the dyes according
to migration distance and agarose gel percentage and type.
ReactionCleanup
SpinProtocol
1051747_HB 15.04.2008 13:24 Uhr Seite 29
ReactionCleanup
VacuumProtocol
30 MinElute Handbook 03/2008
MinElute Reaction Cleanup Kit Protocol
using a vacuum manifold
MinElute columns can also be used on any vacuum manifold with luer connectors (e.g.,
QIAvac 24 Plus or QIAvac 6S with Luer Adapters). The following protocol is designed
to purify double-stranded DNA from all enzymatic reactions e.g., restriction digestion
and labeling resulting in high end-concentrations of DNA (see page 12). Fragments
ranging from 70 bp to 4 kb are purified from enzymes, primers, nucleotides, and salts
using vacuum-driven sample processing.
Important points before starting
■ The yellow color of Buffer ERC indicates a pH ≤7.5.
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ Switch off vacuum between steps to ensure that a consistent, even vacuum is
applied during manipulations.
Procedure
1. Add 300 µl of Buffer ERC to the enzymatic reaction and mix. The maximum volume
of the enzymatic reaction is 100 µl.
If the enzymatic reaction is in a volume of <20 µl, adjust the volume to 20 µl. If
the enzymatic reaction exceeds 100 µl, split the reaction, add 300 µl of Buffer
ERC to each aliquot of the split reaction, and use the appropriate number of
MinElute columns.
2. Check that the color of the mixture is yellow (similar to Buffer ERC without the
enzymatic reaction).
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate,
pH 5.0, and mix. The color of the mixture will turn to yellow.
3. Prepare the vacuum manifold and MinElute columns according to step 3a, 3b, or 3c.
3a. QIAvac 24 Plus (see page 34):
Insert up to 24 MinElute columns into the luer extensions of the QIAvac 24 Plus. Close
unused positions with luer caps and connect QIAvac 24 Plus to a vacuum source.
3b. QIAvac 6S manifold (see page 35):
Open QIAvac 6S lid. Place QIAvac Luer Adapter(s), or blanks to seal unused slots,
into the slots of QIAvac top plate, and close the QIAvac 6S lid. Place the waste
tray inside the QIAvac base, and place the top plate squarely over the base.
Attach the QIAvac 6S to a vacuum source.
Insert each MinElute column into a luer connector on the Luer Adapter(s) in the
manifold. Seal unused luer connectors with plugs provided with the QIAvac Luer
Adapter Set.
1051747_HB 15.04.2008 13:24 Uhr Seite 30
MinElute Handbook 03/2008 31
3c. Other vacuum manifolds: follow the supplier's instructions. Insert each MinElute
column into a luer connector.
4. To bind DNA, load the samples into the MinElute columns by decanting or
pipetting, and apply vacuum. After the samples have passed through the column,
switch off the vacuum source.
For maximum recovery, transfer all traces of sample to the column.
5. To wash, add 750 µl of Buffer PE to each MinElute column and apply vacuum.
6. Transfer each MinElute column to a microcentrifuge tube or the provided 2 ml
collection tubes. Centrifuge for 1 min at ≥≥10,000 x g.
IMPORTANT: This spin is necessary to remove residual ethanol (Buffer PE).
7. Place each MinElute column into a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 10 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center
of each membrane, let the columns stand for 1 min, and then centrifuge.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the center of
the membrane for complete elution of bound DNA. The average eluate volume is
9 µl from 10 µl elution buffer volume.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. When using water, make sure that the pH value is
within this range, and store DNA at –20°C as DNA may degrade in the absence
of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM
Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic
reactions.
9. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to
5 volumes of purified DNA. Mix the solution by pipetting up and down before
loading the gel.
Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and
orange G) that facilitate estimation of DNA migration distance and optimization
of agarose gel run time. Refer to Table 3 (page 15) to identify the dyes according
to migration distance and agarose gel percentage and type.
ReactionCleanup
VacuumProtocol
1051747_HB 15.04.2008 13:24 Uhr Seite 31
32 MinElute Handbook 03/2008
Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may arise. For
more information, see also the Frequently Asked Questions page at our Technical
Support Center: www.qiagen.com/FAQ/FAQList.aspx . The scientists in QIAGEN
Technical Services are always happy to answer any questions you may have about
either the information or protocols in this handbook or sample and assay technologies
(for contact information, see back cover or visit www.qiagen.com ).
Comments and suggestions
Low or no recovery
a) Buffer PE did not Ethanol must be added to Buffer PE (concentrate) before
contain ethanol use. Repeat procedure with correctly prepared Buffer PE.
b) Inappropriate DNA will only be eluted efficiently in the presence of
elution buffer low-salt buffer (e.g., Buffer EB: 10 mM Tris·Cl, pH 8.5)
or water. See “Elution in low-salt solutions”, page 14.
c) Elution buffer Add elution buffer to the center of the MinElute membrane
incorrectly dispensed to ensure that the buffer completely covers the membrane.
d) Binding mixture turns The pH in the sample exceeds the buffer capacity of
orange or violet Buffer PB, QG, or ERC respectively. Add 20 µl of 3 M
sodium acetate, pH 5.0, to the sample and mix. The
color of the mixture will turn yellow indicating the
correct pH for DNA binding. Even for samples with slight
color changes (orange color), add 10 µl sodium acetate.
Gel
e) Gel slice incompletely After addition of Buffer QG to the gel slice, mix by
solubilized vortexing the tube every 2–3 minutes during the 50°C
incubation. DNA will remain in any undissolved agarose.
f) pH of electrophoresis The electrophoresis buffer has been repeatedly used or
buffer too high (binding incorrectly prepared, resulting in a sample pH that
mixture turns orange exceeds the buffering capacity of Buffer QG and leads
or violet) to inefficient DNA binding. Add 10 µl of 3 M sodium
acetate, pH 5.0 to the sample and mix. The color of the
mixture will turn yellow indicating the correct pH for
DNA binding. Even for binding mixtures with only small
color changes (slight orange color), add the 10 µl
sodium acetate.
Gel: refers to MinElute Gel Extraction Kits only; PCR: refers to MinElute PCR Purification Kits only;
Reaction Cleanup: refers to MinElute Reaction Cleanup Kits only; Other notes refer to all kits.
1051747_HB 15.04.2008 13:24 Uhr Seite 32
MinElute Handbook 03/2008 33
Comments and suggestions
g) Cloudy and gelatinous This may be due to salt precipitation, and will disappear
appearance of sample upon mixing the sample.
mixture after addition
of isopropanol
Reaction Cleanup
h) Sample volume too The sample volume must be in the range of 20–100 µl.
high or low
PCR
i) Insufficient/no PCR Estimate DNA recovery by running 10% of PCR product
product before and after purification on an agarose gel.
DNA does not perform well (e.g., in ligation reaction)
a) Salt concentration Modify the wash step by incubating the MinElute column for
in eluate too high 5 min at room temperature after adding 750 µl of
Buffer PE, then centrifuge.
b) Eluate contains residual Ensure that the wash flow-through is drained from the
ethanol collection tube and that the MinElute column is then
centrifuged at ≥10,000 x g for 1 min.
Gel
c) Eluate contaminated The gel slice is incompletely solubilized or weighs
with agarose >400 mg. Be sure to vortex the gel slice in Buffer QG
every 2–3 minutes during the solubilization step.
PCR
d) Eluate contains Primer-dimers are >20 bp, and are not completely
primer-dimers removed. After the binding step, wash the MinElute
column with 750 µl of a 35% (w/v) guanidine
hydrochloride solution (35 g in 100 ml). Continue with the
Buffer PE wash step and the elution step as in the protocol.
e) Eluate contains Use the eluted DNA to prepare the subsequent enzymatic
denatured ssDNA, which reaction but omit the enzyme. To reanneal the ssDNA,
appears as smaller incubate the reaction mixture at 95°C for 2 min, and allow
smeared band on an the tube to cool slowly to room temperature. Add the
analytical gel enzyme and proceed as usual. Alternatively, the DNA can
be eluted in 10 mM Tris buffer containing 10 mM NaCl.
The salt and buffering agent promote the renaturation of
DNA strands. However the salt concentration of the eluate
must then be considered for subsequent applications.
1051747_HB 15.04.2008 13:24 Uhr Seite 33
34 MinElute Handbook 03/2008
Figure 6. QIAvac 24 Plus. Setting up the QIAvac 24 Plus with QIAprep®
, QIAquick®
, MinElute, or RNeasy®
Mini Spin Columns.
1. QIAvac 24 Plus vacuum manifold
2. Luer slot closed with luer plug
3. Spin column*
* Not included with the QIAvac 24 Plus. Included in appropriate purification kits.
QIAGEN1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1
2
3
Appendix: QIAvac Vacuum Manifolds
Handling guidelines for QIAvac 24 Plus
The following guidelines should be followed when working with QIAvac 24 Plus.
■ Always place the QIAvac 24 Plus on a secure bench top or work area. If dropped,
the QIAvac 24 Plus manifold may crack.
■ Always store the QIAvac 24 Plus clean and dry. For cleaning procedures see the
QIAvac 24 Plus Handbook.
■ The components of the QIAvac 24 Plus are not resistant to certain solvents
(Table 4). If these solvents are spilled on the unit, rinse it thoroughly with water.
■ To ensure consistent performance, do not apply silicone or vacuum grease to any
part of the QIAvac 24 Plus manifold.
■ Always use caution and wear safety glasses when working near a vacuum manifold
under pressure.
■ Contact QIAGEN Technical Services or your local distributor for information concerning
spare or replacement parts.
1
2
3
1051747_HB 15.04.2008 13:24 Uhr Seite 34
Handling guidelines for QIAvac 6S
QIAvac 6S facilitates DNA cleanup with the MinElute system by providing a convenient
modular vacuum manifold, which, in combination with QIAvac Luer Adapters, allows
easy processing of MinElute spin columns, as an alternative to centrifugation. The
following recommendations should be followed when handling the QIAvac 6S vacuum
manifold.
■ Always store the QIAvac 6S vacuum manifold clean and dry. To clean, simply
rinse all components with water and dry with paper towels. Do not air-dry, as the
screws may rust and need to be replaced. Do not use abrasives or solvents.
■ Always place the QIAvac 6S vacuum manifold on a secure bench top or work
area. If dropped, the manifold may crack.
■ The components of QIAvac manifolds are not resistant to ethanol, methanol, or
other organic solvents (Table 5). Do not bring solvents into contact with the vacuum
manifold. If solvents are spilled on the unit, rinse thoroughly with distilled water,
and do not incubate acrylic components in alcohol-containing reagents for long
periods of time. Ensure that no residual Buffer PE remains in the vacuum manifold.
■ To ensure consistent performance, do not apply silicone or vacuum grease to any
part of the QIAvac 6S manifold. The spring lock on the top plate and the
self-sealing gasket provide an airtight seal when vacuum is applied to the
assembled unit. To maximize gasket lifetime, rinse the gasket free of salts and
buffers after each use and dry with paper towels before storage.
■ Remove blanks from the slots of the top plate after use and store them under
the manifold.
MinElute Handbook 03/2008 35
Table 4. Chemical Resistance Properties of the QIAvac 24 Plus
Resistant to:
Acetic acid Chaotropic salts Chlorine bleach
Chromic acid Hydrochloric acid SDS
Sodium chloride Sodium hydroxide Tween 20
Urea
Not resistant to:
Benzene Chloroform Ethers
Phenol Toluene
1051747_HB 15.04.2008 13:24 Uhr Seite 35
36 MinElute Handbook 03/2008
Figure 7. QIAvac 6S. Components of the QIAvac 6S manifold.
1. QIAvac base, which holds a waste tray, a strip
holder, or a microtube rack
2. Waste tray
3. QIAvac strip holder to hold 8-well strips
4. QIAvac top plate with slots for 8-well strips or
QIAvac Luer Adapters
5. Microtube rack
6. 8-well strip†
7. Blanks to seal unused slots
8. QIAvac Luer Adapter‡
9. MinElute column
10. Plug to seal unused luer connectors†
10
8
9
5
71
2
4
6
3
6
* MinElute kits are not available in 8-well strip format.
†
Not included with QIAvac 6S. Must be purchased separately.
‡
Not included with QIAvac 6S. Included in appropriate kits.
Table 5. Chemical Resistance Properties of the QIAvac 6S
Resistant to:
Chlorine bleach (12%) Sodium chloride Urea
Hydrochloric acid Sodium hydroxide
Not resistant to:
Acetic acid Acetone Benzene
Chloroform Chromic acid Concentrated alcohol
Ethers Phenol Toluene
1051747_HB 15.04.2008 13:24 Uhr Seite 36
MinElute Handbook 03/2008 37
References
1. Vogelstein, B. and Gillespie, D. (1979) Preparative and analytical purification of
DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615.
2. Hamaguchi, K. and Geiduschek, E.P. (1962) The effect of electrolytes on the
stability of deoxyribonucleate helix. J. Am. Chem. Soc. 84, 1329.
1051747_HB 15.04.2008 13:24 Uhr Seite 37
38 MinElute Handbook 03/2008
Ordering Information
Product Contents Cat. no.
MinElute Kits
MinElute PCR Purification Kit 50 MinElute Spin Columns, 28004
(50) Buffers, Collection Tubes (2 ml)
MinElute PCR Purification Kit 250 MinElute Spin Columns, 28006
(250) Buffers, Collection Tubes (2 ml)
MinElute Gel Extraction Kit 50 MinElute Spin Columns, 28604
(50) Buffers, Collection Tubes (2 ml)
MinElute Gel Extraction Kit 250 MinElute Spin Columns, 28606
(250) Buffers, Collection Tubes (2 ml)
MinElute Reaction Cleanup Kit 50 MinElute Spin Columns, 28204
(50) Buffers, Collection Tubes (2 ml)
MinElute Reaction Cleanup Kit 250 MinElute Spin Columns, 28206
(250) Buffers, Collection Tubes (2 ml)
MinElute 96 UF PCR Purification 4 MinElute 96 UF PCR Purification 28051
Kit (4) Plates
MinElute 96 UF PCR Purification 24 MinElute 96 UF PCR Purification 28053
Kit (24) Plates
Related products
QIAquick PCR Purification 50 QIAquick Spin Columns, Buffers, 28104
Kit (50) Collection Tubes (2 ml)
QIAquick PCR Purification 250 QIAquick Spin Columns, 28106
Kit (250) Buffers, Collection Tubes (2 ml)
QIAquick Nucleotide Removal 50 QIAquick Spin Columns, Buffers, 28304
Kit (50) Collection Tubes (2 ml)
QIAquick Nucleotide Removal 250 QIAquick Spin Columns, 28306
Kit (250) Buffers, Collection Tubes (2 ml)
QIAquick Gel Extraction 50 QIAquick Spin Columns, Buffers, 28704
Kit (50) Collection Tubes (2 ml)
QIAquick Gel Extraction 250 QIAquick Spin Columns, 28706
Kit (250) Buffers, Collection Tubes (2 ml)
1051747_HB 15.04.2008 13:24 Uhr Seite 38
MinElute Handbook 03/2008 39
Ordering Information
Product Contents Cat. no.
Individual Buffers
Buffer PB (500 ml) 500 ml Binding Buffer 19066
Buffer PE (concentrate, 100 ml) 100 ml Wash Buffer 19065
(5x concentrate for 500 ml buffer)
Buffer QG* (250 ml) 250 ml Solubilization and 19063
Binding Buffer (with pH indicator)
QIAvac Manifolds and Accessories
QIAvac 24 Plus Vacuum manifold for processing 19413
1–24 spin columns: includes
QIAvac 24 Plus Vacuum Manifold.
Luer Plugs, Quick Couplings
QIAvac 6S Vacuum manifold for processing 19503
1–24 spin columns: includes Top
Plate with flip-up lid, Base, Waste
Tray, Blanks, Strip Holder, Rack
of Collection Microtubes (1.2 ml)
QIAvac Luer Adapter Set†
For processing 1–24 QIAGEN 19541
spin columns on QIAvac 6S:
6 adapters with 4 luer connectors
each, 24 plugs
Vacuum Pump Universal vacuum pump 84000‡
(capacity 34 L/min, 8 mbar 84010§
vacuum abs.) 84020¶
* Additional Buffer QG may be required for routine purifications from gel slices >300 mg from gels containing
>2% agarose.
†
Compatible only with QIAvac Top Plates containing flip-up lid.
‡
Japan.
§
North America.
¶
Rest of world.
1051747_HB 15.04.2008 13:24 Uhr Seite 39
40 MinElute Handbook 03/2008
Ordering Information
Product Contents Cat. no.
QIAcube and accessories
QIAcube** Robotic workstation for automated 9001292††
purification of DNA, RNA, 9001293¶
or proteins using QIAGEN
spin-column kits, 3-year warranty
on parts and labor
Starter Pack, QIAcube‡‡
Pack includes: reagent bottle racks (3); 990395
rack labeling strips (8); 200 µl
filter-tips (1024); 1000 µl filter-tips
(1024); 1000 µl filter-tips,
wide-bore (1024); 30 ml reagent
bottles (18); rotor adapters (120);
rotor adapter holder
** Agreements for comprehensive service coverage are available; please inquire.
††
US, Canada, and Japan.
‡‡
All starter pack items are available separately.
¶
Rest of world.
1051747_HB 15.04.2008 13:24 Uhr Seite 40
MinElute Handbook 03/2008 41
Notes
1051747_HB 15.04.2008 13:24 Uhr Seite 41
42 MinElute Handbook 03/2008
Notes
1051747_HB 15.04.2008 13:24 Uhr Seite 42
MinElute Handbook 03/2008 43
Trademarks: QIAGEN®
, QIAquick®
, MinElute®
, QIAcube®
, RNeasy®
(QIAGEN Group); Tween®
(ICI Americas Inc.)
Limited License Agreement
Use of this product signifies the agreement of any purchaser or user of the MinElute PCR Purification Kit, the MinElute Gel
extraction Kit and the MinElute Reaction Cleanup Kit to the following terms:
1. The MinElute PCR Purification Kit, the MinElute Gel extraction Kit and the MinElute Reaction Cleanup Kit may be used solely
in accordance with the MinElute Handbook and for use with components contained in the Kit only. QIAGEN grants no
license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components
not included within this Kit except as described in the MinElute Handbook and additional protocols available at
www.qiagen.com .
2. Other than expressly stated licenses, QIAGEN makes no warranty that this Kit and/or its use(s) do not infringe the rights of
third-parties.
3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate
any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall
recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement
or any of its intellectual property rights relating to the Kit and/or its components.
For updated license terms, see www.qiagen.com .
© 2007–2008 QIAGEN, all rights reserved.
1051747_HB 15.04.2008 13:24 Uhr Seite 43
Sample & Assay Technologies
www.qiagen.com
Australia ■ Orders 03-9840-9800 ■ Fax 03-9840-9888 ■ Technical 1-800-243-066
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Korea (South) ■ Orders 1544 7145 ■ Fax 1544 7146 ■ Technical 1544 7145
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1051747 03/2008
1051747_HB 15.04.2008 13:24 Uhr Seite 44
BenchProtocol
Sample & Assay Technologies
Pleasetearhere
Bench Protocol: MinElute PCR Purification
Microcentrifuge and Vacuum Protocol
New users are strongly advised to familiarize themselves with the detailed protocols
and safety information provided in the MinElute Handbook before using this bench
protocol.
Notes before starting
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a
conventional tabletop microcentrifuge at room temperature.
■ Add 1:250 volume pH indicator I to Buffer PB. The yellow color of Buffer PB
with pH indicator I indicates a pH of Յ7.5.
Note: If the purified PCR product is to be used in sensitive microarray
applications, it may be beneficial to use Buffer PB without addition of pH
indicator I. Do not add pH indicator I to buffer aliquots.
Procedure
1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix.
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate,
pH 5.0, and mix. The color of the mixture will turn to yellow.
2. Place a MinElute column in ▲ a provided 2 ml collection tube or into
● a vacuum manifold.
See the MinElute Handbook for details on how to set up a vacuum manifold.
3. To bind DNA, apply the sample to the MinElute column and ▲ centrifuge for
1 min or ● apply vacuum to the manifold until all samples have passed through
the column. ▲ Discard flow-through and place the MinElute column back into
the same tube.
4. To wash, add 0.75 ml Buffer PE to the MinElute column and ▲ centrifuge for
1 min or ● apply vacuum. ▲ Discard flow-through and place the MinElute
column back in the same tube.
5. Centrifuge the column in a 2 ml collection tube (provided) for 1 min.
6. Place each MinElute column in a clean 1.5 ml microcentrifuge tube.
7. To elute DNA, add 10 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the
center of the MinElute membrane, let the column stand for
1 min, and then centrifuge the column for 1 min.
8. If the purified DNA is to be analyzed on a gel, add 1 volume
of Loading Dye to 5 volumes of purified DNA. Mix the
solution by pipetting up and down before loading the gel.
1051747_TearOut 27.02.2008 12:04 Uhr Seite 1
BenchProtocol
Sample & Assay Technologies
Pleasetearhere
Bench Protocol: MinElute Reaction Cleanup
Microcentrifuge and Vacuum Protocol
New users are strongly advised to familiarize themselves with the detailed protocols
and safety information provided in MinElute Handbook before using this bench
protocol.
Notes before starting
■ The yellow color of Buffer ERC indicates a pH Յ7.5.
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ All centrifugation steps are carried out at 10,000 x g in a conventional
table-top microcentrifuge.
Procedure
1. Add 300 µl of Buffer ERC to the enzymatic reaction and mix. Check that the
color of the mixture is yellow (similar to Buffer ERC without dissolved agarose).
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate,
pH 5.0, and mix. The color of the mixture will turn to yellow.
2. Place a MinElute column in ▲ a provided 2 ml collection tube or into
● a vacuum manifold.
See MinElute Handbook for details on how to set up a vacuum manifold.
3. To bind DNA, apply the sample to the MinElute column and ▲ centrifuge for
1 min or ● apply vacuum to the manifold until all samples have passed through
the column. ▲ Discard flow-through and place the MinElute column back into
the same tube.
The maximum volume of the column reservoir is 800 µl. For sample volumes of
more than 800 µl, simply load and spin again.
4. To wash, add 0.75 ml of Buffer PE to MinElute column and ▲ centrifuge for
1 min or ● apply vacuum. ▲ Discard flow-through and place the MinElute
column back into the same tube.
5. Centrifuge the column in a 2 ml collection tube (provided) for 1 min at
10,000 x g.
6. Place MinElute column into a clean 1.5 ml microcentrifuge tube.
7. To elute DNA, add 10 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the
center of the MinElute membrane, let the column stand for
1 min, and then centrifuge the column for 1 min.
8. If the purified DNA is to be analyzed on a gel, add 1 volume
of Loading Dye to 5 volumes of purified DNA. Mix the
solution by pipetting up and down before loading the gel.
1051747_TearOut 27.02.2008 12:05 Uhr Seite 2
BenchProtocol
Sample & Assay Technologies
Pleasetearhere
Bench Protocol: MinElute Gel Extraction
Microcentrifuge and Vacuum Protocol
New users are strongly advised to familiarize themselves with the detailed protocols
and safety information provided in the MinElute Handbook before using this bench
protocol.
Notes before starting
■ The yellow color of Buffer QG indicates a pH Յ7.5.
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ Isopropanol (100%) and a heating block or water bath at 50°C are required.
■ All centrifugation steps are carried out at 10,000 x g in a conventional
table-top microcentrifuge.
Procedure
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume
of gel (100 mg or approximately 100 µl).
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To
help dissolve gel, mix by vortexing the tube every 2–3 min during the
incubation.
For >2% gels, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture
is yellow (similar to Buffer QG without dissolved agarose).
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate,
pH 5.0, and mix. The color of the mixture will turn to yellow.
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a MinElute spin column in ▲ a provided 2 ml collection tube or into
● a vacuum manifold.
See MinElute Handbook for details on how to set up a
vacuum manifold.
1051747_TearOut 27.02.2008 12:05 Uhr Seite 3
BenchProtocol
Sample & Assay Technologies
Pleasetearhere
7. To bind DNA, apply the sample to the MinElute column and ▲ centrifuge for
1 min or ● apply vacuum to the manifold until all samples have passed through
the column. ▲ Discard flow-through and place the MinElute column back into
the same tube.
The maximum volume of the column reservoir is 800 µl. For sample volumes of
more than 800 µl, simply load and spin again.
8. Add 0.5 ml of Buffer QG to the MinElute column and ▲ centrifuge for 1 min or
● apply vacuum. ▲ Discard flow-through and place the MinElute column back
into the same tube.
9. To wash, add 0.75 ml of Buffer PE to MinElute column and ▲ centrifuge for
1 min or ● apply vacuum. ▲ Discard flow-through and place the MinElute
column back into the same tube.
Note: If the DNA will be used for salt sensitive applications, such as blunt-end
ligation and direct sequencing, let the column stand 2–5 min after addition of
Buffer PE, before centrifuging.
10. Centrifuge the column in a 2 ml collection tube (provided) for 1 min at
10,000 x g.
11. Place MinElute column into a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 10 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the
center of the MinElute membrane, let the column stand for 1 min, and then
centrifuge the column for 1 min.
13. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye
to 5 volumes of purified DNA. Mix the solution by pipetting up and down
before loading the gel.
1051747_TearOut 27.02.2008 12:05 Uhr Seite 4