March 2008
QIAquick® Spin Handbook
QIAquick PCR Purification Kit
For purification of PCR products, 100 bp to 10 kb
QIAquick Nucleotide Removal Kit
For oligonucleotide (17-40mers) and DNA
(40 bp to 10 kb) cleanup from enzymatic reactions
QIAquick Gel Extraction Kit
For gel extraction or cleanup of DNA
(70 bp to 10 kb) from enzymatic reactions
••••• ••••• •••••
QIAGEN
Sample & Assay Technologies
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.qiaaen.com .
Contents
Kit Contents 4
Storage 4
Product Use Limitations 5
Product Warranty and Satisfaction Guarantee 5
Quality Control 5
Technical Assistance 5
Safety Information 6
Product Specifications 7
Introduction 8
The QIAquick Principle 11
Equipment and Reagents to Be Supplied by User 18 QIAquick PCR Purification Kit Protocols
I   using a microcentrifuge 19
I   using a vacuum manifold 21
QIAquick Nucleotide Removal Kit Protocol 23 QIAquick Gel Extraction Kit Protocols
I   using a microcentrifuge 25
I   using a vacuum manifold 27
Troubleshooting Guide 30
Appendix: QIAvac Vacuum Manifolds 33
References 36
Ordering Information 37
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Kit Contents
QIAquick PCR Purification Kits	(50)	(250)
Catalog no.	28104	28106
QIAquick Spin Columns	50	250
Buffer PB*	30 ml	150 ml
Buffer PE (concentrate)	2 x 6 ml	55 ml
Buffer EB	15 ml	55 ml
pH Indicator 1	800 ul	800 pi
Collection Tubes (2 ml)	50	250
Loading Dye	110 pi	550 pi
Handbook	1	1
		
QIAquick Nucleotide Removal Kits	(50)	(250)
Catalog no.	28304	28306
QIAquick Spin Columns	50	250
Buffer PN*	30 ml	140 ml
Buffer PE (concentrate)	2 x 6 ml	55 ml
Buffer EB	15 ml	55 ml
Collection Tubes (2 ml)	100	500
Loading Dye	110 pi	550 pi
Handbook	1	1
		
QIAquick Gel Extraction Kits	(50)	(250)
Catalog no.	28704	28706
QIAquick Spin Columns	50	250
Buffer QG*	2x50 ml	2 x 250 ml
Buffer PE (concentrate)	2 x 10 ml	2 x 50 ml
Buffer EB	15 ml	2 x 15 ml
Collection Tubes (2 ml)	50	250
Loading Dye	110 pi	550 pi
Handbook	1	1
* Buffers PB, PN, and QG contain chaotropic salts which are irritants. Take appropriate laboratory safety measures and wear gloves when handling.
Storage
QIAquick Spin Kits should be stored dry at room temperature (15-25°C). Under these conditions, QIAquick Spin 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 the buffers should be redissolved before use. Make sure that all buffers and spin colums are at room temperature when used.
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QIAquick Spin Handbook 03/2008
Product Use Limitations
QIAquick PCR Purification, QIAquick Nucleotide Removal, and QIAquick Gel Extraction Kits are intended for research use. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.
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 or distributor. 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 QIAquick PCR Purification, QIAquick Nucleotide Removal, and QIAquick Gel Extraction Kits is tested against predetermined specifications to ensure consistent product quality.
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 QIAquick Spin Kits, or QIAGEN products in general, please do not hesitate to contact us.
QIAGEN customers are also 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 also 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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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.qiaaen.com/ts/msds.asp where you can find, view, and print the MSDS for each QIAGEN kit and kit component.
Buffer PB contains guanidine hydrochloride, which can form highly reactive compounds when combined with bleach.
In case liquid containing this buffer 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 QIAquick system.
Buffer PB
Contains guanidine hydrochloride and isopropanol: harmful, irritant, flammable. Risk and safety phrases*: Rl0-22-36/38. S23-26-36/37/39-46
Contains sodium Perchlorate and isopropanol: harmful, highly flammable. Risk and safety phrases*: Rl 1-22. SI3-16-23-26-36-46
Contains guanidine thiocyanate: harmful. Risk and safety phrases*: R20/21/22-32. SI 3-26-36-46
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
* RIO: Flammable. Rll: Highly 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. SI 3: Keep away from food, drink and animal feedingstuffs. SI6: Explosive when mixed with oxidizing substances. 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.
A
CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation waste.
Buffer PN
Buffer QG
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QIAquick Spin Handbook 03/2008
Product Specifications
		QIAquick	QIAquick	QIAquick
		PCR Purification	Nucleotide	Gel Extraction
		Kit	Removal Kit	Kit
Maximum binding capacity		10 ug	10 ug	10 ug
Maximum weight of gel slice		—	—	400 mg
Minimum elution volume		30 ul	30 ul	30 ul
Capacity of column reservoir		800 ul	800 ul	800 ul
Typical recoveries				
	Recovery of DNA	90-95%	80-95%	70-80%
		(100 bp - 10 kb)	(40 bp - 10 kb)	(70 bp - 10 kb)
	recovery of oligonucleotides			
	;i7-40mers)	0	60-80%	10-20%
	Recovered			
Oligonucleotides		—	1 7-40mers	—
dsDNA		100 bp - 10 kb	40 bp - 10 kb	70 bp - 10 kb
	Removed			
	<1 Omers	YES	YES	YES
	1 7-40 me rs	YES	no	no
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Introduction
The QIAquick system, designed for rapid DNA cleanup, includes:
■ QIAquick PCR Purification Kits for direct purification of double- or single-stranded PCR products (100 bp - 10 kb) from amplification reactions and DNA cleanup from other enzymatic reactions.
■ QIAquick Nucleotide Removal Kits for general cleanup of oligonucleotides and DNA up to 10 kb from enzymatic reactions (e.g., labeling, dephosphorylation, restriction, and tailing).
■ QIAquick Gel Extraction Kits for extraction of DNA fragments (70 bp - 10 kb) from standard, or low-melt agarose gels in TAE (Tris-acetate/EDTA) or TBE (Tris-borate/ EDTA) buffer and DNA cleanup from enzymatic reactions.
QIAquick PCR Kits are also available in multiwell format for preparation of 8 to 96 samples
(see page 37 for ordering information).
Enzymatic reaction cleanup using QIAquick Kits
The QIAquick system is suitable for fast cleanup of up to 10 ug of DNA fragments from enzymatic reactions and agarose gels (Table 1). Enzyme contamination of DNA samples can interfere with subsequent downstream applications. QIAquick Spin Kits can be used for highly efficient removal of a broad spectrum of enzymes widely used in molecular biology. In addition, QIAGEN offers the MinElute® Reaction Cleanup Kit, which is specially designed for fast and easy DNA cleanup from all enzymatic reactions. Using proven microspin technology, the MinElute Reaction Cleanup Kit delivers highly concentrated purified DNA by using an elution volume of only 10 ul (see ordering information, page 37).
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QIAquick Spin Handbook 03/2008
Table 1. QIAquick DNA Cleanup Guide
		From solutions		From gels
QIAquick PCR Purification Kit		QIAquick Nucleotide Removal Kit	QIAquick Gel Extraction Kit	QIAquick Gel Extraction Kit
Alkaline phosphatase	YES	YES	YES	YES
cDNA synthesis	YES	no	no	YES
DNase,	YES	YES	YES	YES
nuclease digestion				
Kinase:				
DNA fragments	YES	YES	YES	YES
Oligonucleotides	no	YES	no	no
Ligation	YES	YES	YES	YES
Nick translation	YES	YES	YES	YES
PCR	YES	no	no	YES
Random priming	YES	YES	YES	YES
Restriction digestion	YES	YES	YES	YES
Tailing:				
DNA fragments	YES	YES	YES	YES
Oligonucleotides	no	YES	no	no
				
QIAquick Kits provide high yields of pure nucleic acids, for direct use in applications such as:
■ Fluorescent and radioactive sequencing ■    Ligation and transformation
■ Restriction ■ Amplification
■ Labeling ■    In vitro transcription
■ Hybridization ■ Microinjection
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Fragments removed | Fragments recovered
QIAquick PCR Purification Kit
QIAquick Gel Extraction Kit
QIAquick Nucleotide Removal Kit
— — Ji.    £ V
O VI      o   o o
trtr     u- u~ u-
"D -a
"T
§   DNA fragment binding size range
Figure 1. DNA fragment binding-size range. Recoveries of DNA fragments in the size range between "removed" and "recovered" are not defined.
Automated DNA cleanup
The QIAquick PCR Purification Kit and QIAquick 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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QIAquick Spin Handbook 03/2008
The QIAquick Principle
The QIAquick system combines the convenience of spin-column technology with the selective binding properties of a uniquely designed silica membrane. 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). QIAquick spin columns offer 3 handling options — as an alternative to processing the spin columns in a microcentrifuge, they can now also be used on any commercial vacuum manifold with luer connectors (e.g., QIAvac 6S or QIAvac 24 Plus with QIAvac Luer Adapters) or automated on the QIAcube.
Adsorption to QIAquick membrane — salt and pH dependence
The QIAquick silica membrane is uniquely adapted to purify DNA from both aqueous solutions and agarose gels, and up to 10 ug DNA can bind to each QIAquick column. The binding buffers in QIAquick Spin Kits provide the correct salt concentration and pH for adsorption of DNA to the QIAquick 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 95% if the pH is <7.5, and is reduced drastically at higher pH (Figure 1). 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.
lOOn
OH-1-1-1-1-1-1-1
2      4      6      8     10    12 14
PH
Figure 2. pH dependence of DNA adsorption to QIAquick membranes. 1 (Jg 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.
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Optimized binding buffers for ever/ DNA cleanup task
All QIAquick Spin Kits contain identical QIAquick spin columns but different binding buffers optimized for each specific application:
■ Buffer PB in the QIAquick PCR Purification Kit allows the efficient binding of single-or double-stranded PCR products as small as 100 bp and the quantitative (99.5%] removal of primers up to 40 nucleotides. This kit can therefore be used to remove oligo-dT primers after cDNA synthesis or to remove unwanted linkers in cloning experiments.
■ Buffer PN in the QIAquick Nucleotide Removal Kit promotes the adsorption of both oligonucleotides >17 bases and DNA fragments up to 10 kb to the membrane.
■ Buffer QG in the QIAquick Gel Extraction Kit solubilizes the agarose gel slice and provides the appropriate conditions for binding of DNA to the silica membrane.
All of these buffers are available separately (see ordering information, page 37). pH indicator
Binding buffer PB and binding and solubilization buffer QG are specially optimized for use with the QIAquick silica membrane. Buffer QG contains 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 <7.5, 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 or QG 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 QIAquick Gel
Optimal pH pH too high
Figure 3. Indicator enables easy checking of the optimal pH. Indicator dye in solubilization and binding Buffers QG and PB 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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QIAquick Spin Handbook 03/2008
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 and QG do not contain sodium iodide (Nal). Residual Nal may be difficult to remove from DNA samples, and reduces the efficiency of subsequent enzymatic reactions such as blunt-end ligation.
Washing
During the DNA adsorption step, unwanted primers and impurities, such as salts, enzymes, unincorporated nucleotides, agarose, dyes, ethidium bromide, oils, and detergents (e.g., DMSO, 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 50 or 30 ul of the provided Buffer EB (10 mM Tris-CI, pH 8.5), or water. 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-CI, 1 mM EDTA, pH 8.0) is possible, but not recommended because EDTA may inhibit subsequent enzymatic reactions.
DNA yield and concentration
DNA yield depends on the following three factors: the volume of elution buffer, how the buffer is applied to the column, and the incubation time of the buffer on the column. 100-200 ul of elution buffer completely covers the QIAquick membrane, ensuring maximum yield, even when not applied directly to the center of the membrane. Elution with <50 ul requires the buffer to be added directly to the center of the membrane, and if elution is done with the minimum recommended volume of 30 ul, an additional 1 minute incubation is required for optimal yield. DNA will be up to 1.7 times more concentrated if the QIAquick column is incubated for 1 minute with 30 ul of elution buffer, than if it is eluted in 50 ul without incubation (Figure 4, page 14).
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I I I I I
30        40 50        100 200
Elution volume (ul]
Figure 4. Highly concentrated DNA. Effect of elution buffer volume on DNA yield for □ the QIAquick PCR Purification and QIAquick Nucleotide Removal Kit; □ the QIAquick Gel Extraction Kit. 5 ug of a 2.9 kb DNA fragment were purified and eluted with the indicated volumes of Buffer EB. 30 ul plus 1 minute incubation on the QIAquick column gives DNA yields similar to 50 ul without incubation, but at a concentration 1.7 times greater.
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 2 (page 15) 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.
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Table 2. 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 (<50bp)	<50 bp   (<50 bp)
Agarose gel analysis of yield
Yields of DNA following cleanup can be determined by agarose gel analysis. Table 3 shows the total yield obtained following extraction of 1 ug or 0.5 ug starting DNA from an agarose gel with a recovery of 80% or 60% using the QIAquick Gel Extraction Kit. The corresponding amount of DNA in a 1 ul aliquot from 50 ul eluate is indicated. Quantities of DNA fragment corresponding to these 1 ul aliquots are shown on the agarose gel in Figure 4.
Table 3. Amount of DNA in 1 ul aliquots of a 50 ul eluate following QIAquick purification
Starting DNA	Recovery	Total yield	Amount of
		(50 ul eluate)	DNA in 1 ul
1 u9	80%	0.8 ug	16 ng
	60%	0.6 ug	12 ng
0.5 ug	80%	0.4 ug	8 ng
	60%	0.3 ug	6 ng
2.7 kb
Figure 5. High DNA recovery.
Quantities of purified 2.7 kb DNA fragment corresponding to 1 /50 of the DNA obtained following purification from 1 ug or 0.5 ug starting DNA with a recovery of 80% or 60% (see Table 1). Samples were run on a 1 % TAE agarose gel. M: lambda-EcoRI-Hindlll markers.
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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 5).
M    1 25 ng 100 ng 75 ng 50 ng U
Applications using QIAquick purified DNA
DNA purified with QIAquick is suitable for any subsequent application, such as restriction, labeling, hybridization, PCR, ligation and transformation, radioactive and fluorescent sequencing, in vitro transcription, or microinjection.
Figure 6. Agarose gel analysis. An unknown
□ mount 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.
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The QIAquick Procedure
Pure DNA fragment
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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-CI. 1 mM EDTA, pH 8) for elu-tion 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 denaturated alcohol, which contains other substances such as methanol or methylethylketon
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QIAquick PCR Purification Kit Protocol
using a microcentrifuge
This protocol is designed to purify single- or double-stranded DNA fragments from PCR and other enzymatic reactions (see page 8). For cleanup of other enzymatic reactions, follow the protocol as described for PCR samples or use the MinElute Reaction Cleanup Kit. Fragments ranging from 100 bp to 10 kb are purified from primers, nucleotides, polymerases, and salts using QIAquick spin columns in a microcentrifuge.
Important points before starting
■ Add ethanol (96-1 00%) 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 ul pH indicator I to 30 ml Buffer PB or add 600 ul pH indicator I to 150 ml Buffer PB). The yellow color of Buffer PB with pH indicator I indicates a pH of <7.5.
■ 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 sample and mix. It is not necessary to remove mineral oil or kerosene.
For example, add 500 ul of Buffer PB to 100 ul PCR sample (not including oil).
2. If pH indicator I has beein 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 ul of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
3. Place a QIAquick spin column in a provided 2 ml collection tube.
4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60 s.
5. Discard flow-through. Place the QIAquick column back into the same tube.
Collection tubes are re-used to reduce plastic waste.
6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30-60 s.
7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
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8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50 ul Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0-8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 ul elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 ul from 50 ul elution buffer volume, and 28 ul from 30 ul 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 2 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.
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QIAquick Spin Handbook 03/2008
QIAquick PCR Purification Kit Protocol
using a vacuum manifold
QIAquick spin columns can now be used on any vacuum manifold with luer connectors [e.g., QIAvac 6S orQIAvac 24 Plus with Luer Adapters). The following protocol is designed to purify single-or double-stranded DNA fragments from PCR and other enzymatic reactions [see page 8). For cleanup of other enzymatic reactions, follow the protocol as described for PCR samples or use the MinElute Reaction Cleanup Kit. Fragments ranging from 100 bp to 10 kb are purified from primers, nucleotides, polymerases and salts using vacuum-driven sample processing.
Important points before starting
■ Add ethanol (96-1 00%) 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 ul pH indicator I to 30 ml Buffer PB or add 600 ul pH indicator I to 150 ml Buffer PB). The yellow color of Buffer PB with pH indicator I indicates a pH of <7.5.
■ 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 sample and mix. It is not necessary to remove mineral oil or kerosene.
For example, add 500 ul of Buffer PB to 100 ul PCR sample (not including oil).
2. If pH indicator I has beein 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 ul 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 QIAquick columns according to step 3a, 3b, or 3c.
3a. QIAvac 24 Plus (see page 33, and Figure 7):
Insert up to 24 QIAquick 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
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3b. QIAvac 6S manifold (see page 34, and Figure 8):
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 QIAquick 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 QIAquick column into a luer connector.
4. To bind DNA, load the samples into the QIAquick columns by decanting or pipetting, and apply vacuum. After the samples have passed through the column, switch off the vacuum source.
The maximum loading volume of the column is 800 ul. For sample volumes greater than 800 ul simply load again.
5. To wash, add 0.75 ml of Buffer PE to each QIAquick column and apply vacuum.
6. Transfer each QIAquick column to a microcentrifuge tube or the provided 2 ml collection tubes. Centrifuge for 1 min at 17,900 x g (13,000 rpm).
IMPORTANT: This spin is necessary to remove residual ethanol (Buffer PE).
7. Place each QIAquick column into a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 50 ul of Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0-8.5) to the center of each QIAquick membrane, and centrifuge the columns for 1 min at 17,900 x g (13,000 rpm). Alternatively, for increased DNA concentration, add 30 ul elution buffer to the center of each QIAquick membrane, let the columns stand for 1 min, and then centrifuge.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 ul from 50 ul elution buffer volume, and 28 ul from 30 ul 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 (10 mM Tris-Cl, 1 mMEDTA, 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 2 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.
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QIAquick Spin Handbook 03/2008
QIAquick Nucleotide Removal Kit Protocol
using a microcentrifuge
This protocol is designed for cleanup of radioactive-, biotin-, or DIG-labeled DNA fragments and oligonucleotides >17 nucleotides from enzymatic reactions (see page 8). The protocol ensures removal of primers <10 bases, enzymes, salts, and unincorporated nucleotides. It is possible to use this kit with a vacuum manifold as well as with a microcentrifuge, and a protocol for vacuum processing is available on request from QIAGEN Technical Services or your local distributor. However, we do not recommend processing radioactive samples with a vacuum manifold.
Important points before starting
■ Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).
■ All centrifugation steps are in a conventional tabletop microcentrifuge at room temperature.
Procedure
1. Add 10 volumes of Buffer PN to 1 volume of the reaction sample and mix.
For example, add 500 ul Buffer PN to a 50 ul reaction sample. For DNA fragments >100 bp, only 5 volumes of Buffer PN are required.
2. Place a QIAquick spin column in a provided 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min at 6000 rpm.
4. For radioactive samples:
Place the QIAquick column into a clean 2 ml collection tube and discard the tube containing the radioactive flow-through appropriately.
For non-radioactive samples:
Discard the flow-through and place QIAquick column back into the same tube.
Collection tubes are reused to reduce plastic waste.
5. For radioactive samples:
To wash QIAquick column, add 500 ul of Buffer PE and centrifuge for 1 min at 6000 rpm. Discard the flow-through appropriately and repeat wash with another 500 ul of Buffer PE.
For non-radioactive samples:
To wash QIAquick column, add 750 ul of Buffer PE and centrifuge for 1 min at 6000 rpm.
QIAquick Spin Handbook 03/2008
23
6. Discard the flow-through and place the QIAquick column back in the same tube, which should be empty. Centrifuge for an additional 1 min at 13,000 rpm (17,900 x g).
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifuge.
7. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 100-200 ul of Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0-8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min at 13,000 rpm (17,900 x g). Alternatively, for increased DNA concentration, add 30-50 ul elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA.
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 (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 2 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.
24
QIAquick Spin Handbook 03/2008
QIAquick Gel Extraction Kit Protocol
using a microcentrifuge
This protocol is designed to extract and purify DNA of 70 bp to 10 kb from standard or low-melt agarose gels in TAE or TBE buffer. Up to 400 mg agarose can be processed per spin column. This kit can also be used for DNA cleanup from enzymatic reactions (see page 8). For DNA cleanup from enzymatic reactions using this protocol, add 3 volumes of Buffer QG and 1 volume of isopropanol to the reaction, mix, and proceed with step 6 of the protocol. Alternatively, use the MinElute Reaction Cleanup Kit.
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 1 7,900 x g (1 3,000 rpm) 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 ~ 100 ul).
For example, add 300 ul 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 QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick 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).
If the color of the mixture is orange or violet, add 10 ul 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 QIAquick 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.
For example, if the agarose gel slice is 100 mg, add 100 ul isopropanol. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage.
QIAquick Spin Handbook 03/2008
25
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
The maximum volume of the column reservoir is 800 ul. For sample volumes of more than 800 ul, simply load and spin again.
8. Discard flow-through and place QIAquick column back in the same collection tube.
Collection tubes are reused to reduce plastic waste.
9. Recommended: Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
This step will remove all traces of agarose. It is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription, or microinjection.
10. To wash, add 0.75 ml of Buffer PE to QIAquick 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.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13. To elute DNA, add 50 ul of Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0-8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 ul elution buffer to the center of the QIAquick 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 QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 ul from 50 ul elution buffer volume, and 28 ul from 30 ul.
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 (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
14. 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 2 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.
26
QIAquick Spin Handbook 03/2008
QIAquick Gel Extraction Kit Protocol
using a vacuum manifold
QIAquick spin columns can now be used on any vacuum manifold with luer connectors [e.g., QIAvac 6S or QIAvac 24 Plus with Luer Adapters). The following protocol is designed to extract and purify DNA of 70 bp to 10 kb from standard or low-melt agarose gels in TAE or TBE buffer using vacuum-driven processing. Up to 400 mg agarose can be processed per spin column. This kit can also be used for DNA cleanup from enzymatic reactions (see page 8). For DNA cleanup from enzymatic reactions using this protocol, add 3 volumes of Buffer QG and 1 volume of isopropanol to the reaction and mix. Set up the vacuum manifold as described in step 4 and then and proceed with step 7 of the protocol. Alternatively, use the new MinElute Reaction Cleanup Kit.
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 ul).
For example, add 300 ul 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 QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick 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 QIAquick columns according to steps 4a, 4b, or 4c.
4a. QIAvac 24 Plus (see page 33, and Figure 7):
Insert up to 24 QIAquick 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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27
4b. QIAvac 6S manifold (see page 34, and Figure 8):
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 QIAquick 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 QIAquick-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 ul 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 QIAquick membrane is efficient only at pH <7.5. Buffer QG contains a pH indicator that 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 ul isopropanol. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage.
7. To bind DNA, pipet the sample onto the QIAquick column and apply vacuum. After the sample has passed through the column, switch off vacuum source.
The maximum volume of the column reservoir is 800 ul. For sample volumes of more than 800 ul, simply load again.
8. Recommended: Add 0.5 ml of Buffer QG to QIAquick column and apply vacuum.
This step will remove all traces of agarose. It is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription, or microinjection.
9. To wash, add 0.75 ml of Buffer PE to QIAquick 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.
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QIAquick Spin Handbook 03/2008
10. Transfer QIAquick column to a clean 1.5 ml microcentrifuge tube or to a provided 2 ml collection tube. Centrifuge for 1 min at 17,900 x g (13,000 rpm).
IMPORTANT: This spin is necessary to remove residual ethanol (Buffer PE).
11. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 50 ul of Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7-8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min at 17,900 x g (13,000 rpm). Alternatively, for increased DNA concentration, add 30 ul elution buffer, let stand for 1 min, and then centrifuge for 1 min.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 ul from 50 ul elution buffer volume, and 28 ul from 30 ul.
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 2 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.
Vacui	Gel
3	m X
Protocc	fraction
	
QIAquick Spin Handbook 03/2008
29
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 contain ethanol
b) Inappropriate elution buffer
c) Elution buffer incorrectly dispensed
Gel
d) Gel slice incompletely solubilized
e) pH of electrophoresis buffer too high (binding mixture turns orange or violet]
Gel slice was too large (>400 mg]
Ethanol must be added to Buffer PE (concentrate) before use. Repeat procedure with correctly prepared Buffer PE.
DNA will only be eluted efficiently in the presence of low-salt buffer (e.g., Buffer EB: 10 mMTris-CI, pH 8.5) or water. See "Elution in low-salt solutions", page 13.
Add elution buffer to the center of the QIAquick membrane to ensure that the buffer completely covers the membrane. This is particularly important when using small elution volumes (30 ul).
After addition of Buffer QG to the gel slice, mix by vortexing the tube every 2-3 min during the 50°C incubation. DNA will remain in any undissolved agarose.
The electrophoresis buffer has been repeatedly used or incorrectly prepared, resulting in a sample pH that exceeds the buffering capacity of Buffer QG and leads to inefficient DNA binding. Add 10 ul 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 ul sodium acetate.
70-80% recovery can only be obtained from <400 mg gel slice per QIAquick column. For gel slices >400 mg, use multiple QIAquick columns.
Gel: refers to QIAquick Gel Extraction Kits only. PCR: refers to QIAquick PCR Purification Kits only. Other notes refer to all kits.
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QIAquick Spin Handbook 03/2008
Comments and Suggestions
PCR
g) Insufficient/no PCR product
PCR/Gel
h) Cloudy and gelatinous appearance of sample mixture after addition of isopropanol
i) Binding mixture turns orange or violet
DNA does not perform well
a) Salt concentration in eluate too high
b) Eluate contains residual ethanol
Gel
c) Eluate contaminated with agarose
PCR
d) Eluate contains primer-dimers
Estimate DNA recovery by running 10% of PCR product before and after purification on an agarose gel.
This may be due to salt precipitation, and will disappear upon mixing the sample. Alternatively, the gel slice may not be completely solubilized. In this case, apply the mixture to the QIAquick column, centrifuge, and then add 0.5 ml Buffer QG to the column. Let stand for 1 min at room temperature, and then centrifuge and continue with the procedure. This additional wash will solubilize remaining agarose.
The pH in the sample exceeds the buffer capacity of Buffer QG or PB respectively. Add 20 ul 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 ul sodium acetate.
[e.g., in ligation reactions)
Modify the wash step by incubating the column for 5 min at room temperature after adding 750 ul of Buffer PE, then centrifuge.
Ensure that the wash flow-through is drained from the collection tube and that the QIAquick column is then centrifuged at 1 7,900 x g (1 3,000 rpm) for an additional 1 min.
The gel slice is incompletely solubilized or weighs >400 mg. Repeat procedure, including the optional Buffer QG column-wash step.
Primer-dimers formed are >20 bp and are not completely removed. After the binding step, wash the QIAquick column with 750 ul of a 35% guanidine hydrochloride aqueous solution (35 g in 100 ml). Continue with the Buffer PE wash step and the elution step as in the protocol.
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31
Comments and Suggestions
e)    Eluate contains denatured ssDNA, which appears as smaller smeared band on an analytical gel
Use the eluted DNA to prepare the subsequent enzymatic reaction but omit the enzyme. To reanneal the ssDNA, incubate the reaction mixture at 95°C for 2 min, and allow the tube to cool slowly to room temperature. Add the enzyme and proceed as usual. Alternatively, the DNA can be eluted in 10 mM Tris buffer containing 10 mM NaCI. 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.
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QIAquick Spin Handbook 03/2008
Appendix: QIAvac Vacuum Manifolds Handling guidelines for 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.
Figure 7. Qlvac 24 Plus. Setting up the QIAvac 24 Plus with QIAprep®, QIAquick, MinElute, or RNeasy8 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.
QIAquick Spin Handbook 03/2008
33
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 Phenol	Chloroform Toluene	Ethers
Handling guidelines for QIAvac 6S
QIAvac 6S facilitates DNA cleanup with QIAquick by providing a convenient modular vacuum manifold, which, in combination with QIAvac Luer Adapters, allows easy processing of QIAquick 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.
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QIAquick Spin Handbook 03/2008
Figure 8. 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 Adapter1
9. QIAquick spin column*
10. Plug to seal unused luer connectors1
Not included with QIAvac Manifold. Included in appropriate kits. Not included with QIAvac 6S. Must be purchased separately.
Table 5. Chemical Resistance Properties of the QIAvac OS
Resistant to:		
Chlorine bleach (12%]	Diluted alcohol	Hydrochloric acid
Sodium chloride	Sodium hydroxide	Urea
Not resistant to:		
Acetone Chromic acid Toluene	Benzene Ethers	Chloroform Phenol
QIAquick Spin Handbook 03/2008
35
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 deoxyribonucle-ate helix. J. Am. Chem. Soc. 84, 1 329.
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QIAquick Spin Handbook 03/2008
Ordering Information
Product	Contents	Cat. no.
QIAquick Spin Kits		
QIAquick PCR Purification Kit (50]	50 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml]	28104
QIAquick PCR Purification Kit (250]	250 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml]	28106
QIAquick Nucleotide Removal Kit (50]	50 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml]	28304
QIAquick Nucleotide Removal Kit (250]	250 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml]	28306
QIAquick Gel Extraction Kit (50]	50 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml]	28704
QIAquick Gel Extraction Kit (250]	250 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml]	28706
Related products		
MinElute Reaction Cleanup Kit (50]	50 MinElute Spin Columns, Buffers, Collection Tubes (2 ml]	28204
MinElute Reaction Cleanup Kit (250]	250 MinElute Spin Columns, Buffers, Collection Tubes (2 ml]	28206
MinElute Gel Extraction Kit (50]	50 MinElute Spin Columns, Buffers, Collection Tubes (2 ml]	28604
MinElute Gel Extraction Kit (250]	250 MinElute Spin Columns, Buffers, Collection Tubes (2 ml]	28606
MinElute PCR Purification Kit (50]	50 MinElute Spin Columns, Buffers, Collection Tubes (2 ml]	28004
MinElute PCR Purification Kit (250]	250 MinElute Spin Columns, Buffers, Collection Tubes (2 ml]	28006
Individual buffers		
Buffer PB (500 ml]	500 ml Binding Buffer	19066
Buffer PN	500 ml Binding Buffer	19071
Buffer PE (concentrate]	100 ml Buffer PE (5x concentrate; final volume 500 ml]	19065
Buffer QG* (250 ml]	250 ml Solubilization and Binding Buffer (with pH indicator]	19063
*   Additional Buffer QG may be required for routine purifications from gel slices >300 mg from gels containing >2% agarose.
QIAquick Spin Handbook 03/2008
37
Ordering Information
Product	Contents	Cat. no.
QIAcube and accessories		
QIAcube*	Robotic workstation for automated	9001292f
	purification of DNA, RNA, or proteins	9001293'
	using QAGEN spin-column kits, 3-year	
	warranty on parts and labor	
Starter Pack, QAcube1	Pack includes: reagent bottle racks (3); rack labeling strips (8); 200 ul filter-tips (1024]; 1000 ul filter-tips (1024); 1000 ul filter-tips, wide-bore (1024]; 30 ml reagent bottles (1 8); rotor adapters (1 20]; rotor adapter holder	990395
QIAvac manifolds and accessories		
QAvac 24 Plus	Vacuum manifold for processing 1-24 spin columns: includes QAvac 24 Plus Vacuum Manifold, Luer Plugs, Quick Couplings	19413
QAvac 6S	Vacuum manifold for processing 1-6 QAGEN 8-well strips: includes QAvac 6S Top Plate with flip-up lid, Base, Waste Tray, Blanks, Strip Holder	19503
QAvac 96	Vacuum manifold for processing QAGEN 96 well-plates: includes QAvac 96 Top plate, Base, Waste Tray, Plate Holder	19504
QAvac Luer Adapter Set1	For processing 1-24 QAGEN spin columns on QAvac 6S: 6 adapters with 4 luer connectors each, 24 plugs	19541
Vacuum Regulator	For use with QAvac manifolds	19530
Vacuum Pump	Universal vacuum pump	84000**
	(capacity 34 L/min, 8 mbar	84010"
	vacuum abs.]	84020"
* Agreements for conprehensive service coverage are available; please inquire.     T US, Canada, and Japan. t Rest of world.     § All starter pack items are available separately.     11 Compatible only with QIAvac Top Plates containing flip-up lid.     ** Japan.     n US and Canada.     w Rest of world.
The QIAcube is intended to be used only in combination with QIAGEN Kits for applications described in the respective Kit handbooks. All other Kits are intended for research use. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.
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QIAquick Spin Handbook 03/2008
Trademarks: QIAGEN®, GIAEX®, QIAquick®, QIAprep®, 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 QIAquick PCR Purification Kit, the QIAquick Nucleotide Removal Kit and the QIAquick Gel extraction Kit to the following terms:
1. The QIAquick PCR Purification Kit, the QIAquick Nucleotide Removal Kit and the QIAquick Gel extraction Kit may be used solely in accordance with the QIAquick Spin 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 QIAquick Spin Handbook and additional protocols available at www.qiaqen.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.
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051746 03/2008
Sample & Assay Technologies
BenchProtocol
Bench Protocol: QIAquick PCR Purification Microcentrifuge and Vacuum Protocol
New users are strongly advised to familiarize themselves with the detailed protocols and safety information provided in the QIAquick Spin 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 <7.5.
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 ul of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
2. Place a QIAquick column in ▲ a provided 2 ml collection tube or into • a vacuum manifold.
See the QIAquick Spin Handbook for details on how to set up a vacuum manifold.
3. To bind DNA, apply the sample to the QIAquick column and ▲ centrifuge for 30-60 s or • apply vacuum to the manifold until all samples have passed through the column. ▲ Discard flow-through and place the QIAquick column back into the same tube.
4. To wash, add 0.75 ml Buffer PE to the QIAquick column and ▲ centrifuge for 30-60 s or • apply vacuum. ▲ Discard flow-through and place the QIAquick column back in the same tube.
5. Centrifuge the column in a 2 ml collection tube (provided) for 1 min.
6. Place each QIAquick column in a clean 1.5 ml microcentrifuge tube.
7. To elute DNA, add 50 ul Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 ul elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.
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.
QIAGEN
Sample & Assay Technologies
Bench Protocol
Bench Protocol: QIAquick Nucleotide Removal Protocol
New users are strongly advised to familiarize themselves with the detailed protocols and safety information provided in the QIAquick Spin 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 in a conventional in a conventional tabletop microcentrifuge.
Procedure
1. Add 10 volumes of Buffer PN to 1 volume of the reaction sample and mix.
2. Place a QIAquick spin column in a provided 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min at 6000 rpm.
4. For radioactive samples:
Place the QIAquick column into a clean 2 ml collection tube and discard the tube containing the radioactive flow-through appropriately. For non-radioactive samples:
Discard the flow-through and place QIAquick column back into the same tube.
5. For radioactive samples:
To wash QIAquick column, add 500 ul of Buffer PE and centrifuge for 1 min at 6000 rpm. Discard the flow-through appropriately and repeat wash with another 500 ul of Buffer PE. For non-radioactive samples:
To wash QIAquick column, add 750 ul of Buffer PE and centrifuge for 1 min at 6000 rpm.
6. Discard the flow-through and place the QIAquick column back in the same tube, which should be empty. Centrifuge for an additional 1 min at 13,000 rpm (17,900 xg).
7. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 100-200 ul of Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min at 13,000 rpm (17,900 x g). Alternatively, for increased DNA concentration, add 30-50 ul elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.
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.
QIAGEN
Sample & Assay Technologies
BenchProtocol
Bench Protocol: QIAquick Gel Extraction Microcentrifuge and Vacuum Protocol
New users are strongly advised to familiarize themselves with the detailed protocols and safety information provided in the QIAquick Spin Handbook before using this bench protocol.
Notes 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).
■ Isopropanol (100%) and a heating block or water bath at 50°C are required.
■ All centrifugation steps are carried out at 17,900 x g (13,000 rpm) 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 - 100 ul).
If the color of the mixture is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
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 ul 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 QIAquick spin column in ▲ a provided 2 ml collection tube or into • a vacuum manifold.
See QIAquick Spin Handbook for details on how to set up a vacuum manifold.
QIAGEN
Sample & Assay Technologies
Bench Protocol
7. To bind DNA, apply the sample to the QIAquick 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 QIAquick column back into Z the same tube. J
The maximum volume of the column reservoir is 800 ul. For sample volumes of more than 800 ul, simply load and spin/apply vacuum again.
8. Recommended: Add 0.5 ml of Buffer QG to QIAquick column and ▲ centrifuge for 1 min or • apply vacuum. ▲ Discard flow-through and place the QIAquick column back into the same tube.
This step is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription, or microinjection.
9. To wash, add 0.75 ml of Buffer PE to QIAquick column and ▲ centrifuge for 1 min or • apply vacuum. ▲ Discard flow-through and place the QIAquick 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 17,900 xg (13,000 rpm).
11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 50 ul of Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 ul elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge 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.
QIAGEN
Sample & Assay Technologies