Genomic DNA

Purification
Technical hints,
applications,
and protocols
 Trademarks and disclaimers

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trademarks of the QIAGEN Group: QIAGEN®, AirPore™, DNeasy®,
Masscode™, QuantiTect™.

ABI PRISM is a registered trademark of Applera Corporation or its

subsidiaries.

Black Hole Quencher and BHQ are trademarks of Biosearch

Technologies, Inc. (BTI).

Triton is a registered trademark of Rohm & Haas Inc.

The PCR process is covered by U.S. Patents 4,683,195 and 4,683,202

and foreign equivalents owned by Hoffmann-La Roche AG.

Purchase of QIAGEN products for PCR is accompanied by a limited

license to use them in the Polymerase Chain Reaction (PCR) process for
research and development activities in conjunction with a thermal cycler
whose use in the automated performance of the PCR process is covered
by the up-front license fee, either by payment to Applied Biosystems or
as purchased, i.e. an authorized thermal cycler. The PCR process is
covered by U.S. Patents 4,683,195 and 4,683,202 and foreign
equivalents owned by Hoffmann-La Roche AG.

The Black Hole Quenchers and BHQ dyes were developed by and
licensed from Biosearch Technologies, Novato, CA. These products are
sold exclusively for R&D use by the purchaser. They may not be used for
clinical or diagnostic purposes and they may not be re-sold, distributed,
or re-packaged.

© 2002 QIAGEN, all rights reserved.

2 Genomic DNA Purification

Contents

Introduction 4
Effects of contaminants on downstream applications 4

Starting materials 5
Sample collection and storage before isolation
of genomic DNA 6
Animal tissue 6
Animal, yeast, and bacterial cell cultures 6
Animal blood 7
Amount of starting material 7
DNA yields 7

Disruption and lysis 8

Disruption methods 8
Enzymatic lysis of animal tissue 8
Enzymatic lysis of bacteria and yeast 8
Disruption using rotor-stator homogenizers 8
Disruption using the Mixer Mill MM 300 and 9
other bead mills
Disruption using a mortar and pestle 9

DNA isolation methods 10

Preparation of crude lysates 10
Salting-out methods 11
Organic extraction methods 11
Cesium chloride density gradients 11
Anion-exchange methods 12
Silica-based methods — DNeasy Tissue Kits 12

Applications using DNeasy Tissue Kits 14

Functional genomics 14
Molecular breeding 16
Genetic studies in bacteria and yeast 17
Disease research 18
Natural history applications 19
Phylogeny and biodiversity studies 20
Detection of viral pathogens 21

Protocols 22

User-Developed Protocols 27

References 29

Ordering Information 30

Genomic DNA Purification 3

 DNA Contamination Leads to Lower Yields Introduction
and Quality of PCR Products
Over the last decade there has been an increased requirement for
sy
isolation of pure genomic DNA that performs well in any down-

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Phenol SDS stream application. Such downstream applications include PCR
ea

ea
DN

M 0.2 0.5%
DN
M 0.005 0.01%
(multiplex PCR, real-time PCR etc.), Southern blotting, AFLP, RFLP,
microsatellite analysis, SNP analysis, and Masscode™ technology.
With the expansion of genomic analysis research, sample sources
are becoming increasingly diverse. Each has its own difficulties
associated with the isolation of pure genomic DNA.

For many analysis techniques, DNA quality is the single most

important factor. Poor quality DNA can lead to suboptimal results,
and DNA that is impure or contaminated will not perform well in
downstream applications. The choice of sample preparation method
directly affects the results of the downstream application.

QIAGEN offers extensive expertise in the purification of nucleic

sy

sy

NaCl Ethanol acids. This guide gives an overview of the techniques used for isolation
ea

ea
DN

DN

M 25 50 mM
M 1 5%
of DNA from a wide variety of animal tissue and cell sources, as
well as guidelines for successful downstream applications. Further
general information on the handling and analysis of genomic DNA is
available in the QIAGEN® Bench Guide. QIAGEN also offers guides
for plant tissues and clinical samples. Please see the brochure Plant
Nucleic Acid Purification, for plants and fungi, and the Application
and Product Guide for Molecular Diagnostics, for human clinical
samples. All QIAGEN literature is available free on request from
QIAGEN Technical Services, or can be downloaded from our web
site: www.qiagen.com/literature/brochures/index.asp.

Figure 1. PCR amplification of genomic DNA purified

using the DNeasy Tissue Kit in the absence (DNeasy) and
presence of increasing concentrations of indicated
impurities shows that impure DNA preparations lead to
failure of PCR and/or low yields of amplicons. From top
left: Phenol: 0.2%, 0.5% (v/v); SDS: 0.005%, 0.01%
(w/v); NaCl: 25 mM, 50 mM; Ethanol: 1%,
5% (v/v); M: markers.
Effects of contaminants on downstream applications
Carryover of contaminants such as salts, phenol, ethanol, and
detergents from conventional purification procedures can inhibit
performance of DNA in downstream applications. DNA prepared
using DNeasy® technology is free from contaminants, ensuring
consistently good performance in downstream applications
(Figures 1 and 2).

Researchers require reliable purification methods that minimize the

risk of DNA contamination, facilitating high-quality results in
downstream applications. There is also a need for purification
methods to be fast, safe, robust, and easy to handle. Many
researchers also want the added benefit of being able to use one
commercial kit for many sample types.

Home-made methods do not fulfill all the above requirements. In

addition, they often adversely affect reproducibility, whereas
commercial kits offer a proven and guaranteed solution. DNeasy
Tissue Kits fulfill all the above purification requirements and
guarantee high-quality DNA with every prep.

4 Genomic DNA Purification

Starting materials Effect of Contamination on Spectrophotometry

Genomic DNA can be isolated from a wide variety of starting 0.4

DNA +
materials, for many different fields of study. These include: phenol (P)

◆ Cultured animal cells

Absorbance
DNA (Q)
◆ Animal tissues (e.g., brain, liver, spleen, lung, heart, and kidney)
◆ Crude lysates of any animal tissue
◆ Animal blood
◆ Buffy coats (from animal blood)
0.0
◆ Animal bone marrow 220 Wavelength (nm) 320

◆ Fixed tissues
Figure 2. Effect of phenol on DNA quantification. UV scan
◆ Yeast of Q (QIAGEN): NA purified using silica-gel–membrane
◆ Bacteria technology (100 µl sample); P (phenol): The same sample
with 1 µl phenol diluted 1:1000 in water (final dilution
◆ Insects
1:100,000). Phenol contamination of DNA can interfere
◆ Rodent tails with spectrophotometric readings and lead to over-
estimation.
DNA is a relatively stable molecule. However, introduction of
enzymatically active nucleases to DNA solutions should be avoided,
as these enzymes will degrade DNA.

DNA is subject to acid hydrolysis when stored in water, and should

therefore be stored at a slightly alkaline pH, e.g., in TE buffer or in
Buffer AE from QIAGEN.

Degradation of DNA has a major effect on any results obtained,

generating errors that are both quantitative and qualitative. For
example, a reduction in DNA size may lead to the failure of
downstream applications such as PCR-based applications and
hybridization. This is especially important in areas where DNA
degradation is a common phenomenon in the original samples,
e.g., forensics or natural history studies.

In this section, QIAGEN offers some hints and tips on the collection
and storage of tissue samples before DNA isolation. For plant tissues
and clinical samples, please see the brochures Plant Nucleic Acid
Purification, and the Application and Product Guide for Molecular
Diagnostics. Both brochures are available free on request.

Genomic DNA Purification 5

Table 1. Maximum recommended amounts of
starting material*
Animal tissue
Mouse tail
Rat tail
Cultured cells
Bacteria
Yeast
* Using DNeasy Tissue procedures.
Table 2. Sizes and molecular weights of
various genomic DNAs
Organism
Escherichia coli K12
Saccharomyces cerevisiae
Dictyostelium discoideum
Arabidopsis thaliana
Caenorhabditis elegans
Drosophila melanogaster
Gallus domesticus (chicken)
Mus musculus (mouse)
Rattus norvegicus (rat)
Xenopus Iaevis
Homo sapiens
6 Genomic DNA Purification
Sample collection and storage before isolation of genomic DNA
The quality of the starting material affects the quality and yield of the
25 mg isolated DNA. Optimal results are obtained with fresh material, or
with material that has been immediately frozen (frozen in liquid
0.6–1.2 cm
nitrogen or in a mixture of ethanol and dry ice) and stored at –20°C
0.6 cm or –70°C. Repeated freezing and thawing of stored samples should
5 x 106 cells be avoided, as this leads to reduced fragment size and precipitation
2 x 109 cells of the DNA, and in clinical samples, to reduced yields of pathogen
5 x 107 cells DNA (e.g., viral DNA). Use of poor quality starting material will also
lead to reduced length and yield of purified DNA. In general,
genomic DNA yields will decrease if samples are stored at either
2–8°C or –20°C without previous treatment.
The recommendations for storage of different starting materials are
discussed below. In some cases, storage under optimal conditions
may not be possible, e.g., for preserved tissues. In such cases the
downstream application may be adjusted to compensate for poor or
inappropriate storage conditions.
Animal tissue
Freshly harvested tissue can be immediately frozen and stored at
–20°C, –70°C, or in liquid nitrogen. Tissue samples can also be
stored in Buffer ATL, after proteinase K digestion, for up to 6 months
at ambient temperatures without any reduction in DNA quality.
Animal and human tissues can also be fixed for storage. We
recommend using fixatives such as alcohol and formalin; however,
long-term storage of tissues in formalin will result in chemical
modification of the DNA, which decreases DNA quality and results
Base pairs per
haploid genome in shorter fragments upon purification. Fixatives that cause cross-
linking, such as osmic acid, are not recommended if DNA will be
4.6 x 106
isolated from the tissue. It is also possible to isolate DNA from
1.2 x 107
paraffin-embedded tissue.
5.4 x 107
1.2 x 108
Animal, yeast, and bacterial cell cultures
9.7 x 107
1.8 x 108 Cell cultures are usually harvested by centrifugation followed by
1.2 x 109 removal of the supernatant. Cells are then stored at –20°C or –70°C.
3.0 x 109 Alternatively, animal cell nuclei can be prepared and stored at
3.0 x 109 –20°C. For certain bacterial and yeast cultures that accumulate large
3.1 x 109 amounts of metabolites and/or form very dense cell walls, cells
3.3 x 109 should be harvested in the early log phase of growth, and washed
with fresh medium. Fresh or frozen cell pellets can be used.
Animal blood Table 3. Yields of genomic DNA with DNeasy
Tissue Kits
Freshly drawn animal whole blood (nucleated or non-nucleated)
should be digested with proteinase K. The amount of blood used is Yield
critical and tenfold less nucleated blood should be used in the Total nucleic
procedure compared with non-nucleated blood. Neither type of Source acids (µg)* DNA (µg)†
blood should be coagulated. Buffer AL should be added to blood Lymphocytes 20–30 15–25
containing proteinase K and incubated for 10 minutes at 70°C for (5 x 106)
digestion. The appropriate DNeasy Tissue Kit protocol should then be
HeLa cells 40–60 15–25
followed.
(2 x 106)

Amount of starting material Liver (25 mg) 60–115 10–30

Brain (25 mg) 35–60 15–30

The amount of starting material to use is a key point in the successful
isolation of genomic DNA. Starting sample size depends on the Lung (25 mg) 8–20 5–10
tissue or cells being used, but the amounts shown in Table 1 (page 6) Heart (25 mg) 25–45 5–10
are a good guide when using DNeasy Tissue procedures. Often, an
Kidney (25 mg) 40–85 15–30
excess of starting material can be counter-productive, and as a gen-
eral guide “less is more”. If you have no information on DNA content Spleen (10 mg) 25–45 5–30
and your starting material is not shown in Table 1, we recommend Mouse tail, 1.2 cm 15–30 10–25
beginning with half the maximum amount of starting material indicat- (tip section)
ed in Table 1. Depending on the yield obtained, the sample size can
Rat tail, 0.6 cm 25–60 20–40
be increased in subsequent preparations.
(tip section)

DNA yields * Nucleic acids purified without RNase treatment.

†
Nucleic acids purified with RNase treatment.
The DNA content of cells is not homogeneous, and depends on the
type of cell and the size of the genome. Genome sizes can vary by
a factor of 1000, e.g., between bacterial (106 bp) and animal
(109 bp) cells (Table 2, page 6). Animal cells usually have genomes
sized between 108 (worms and some insects) and 8 x 1010 bp per cell.

As a guide, one mammalian cell contains an average of 6 pg DNA.

However, some tissues have a very high cell density and therefore
more DNA per milligram of tissue can be expected. For samples with
very high DNA content e.g., spleen or cell lines with a high degree
of ploidy, use less than the recommended amount of starting material
listed in Table 1. Depending on the yield obtained, the sample size
can be increased in subsequent preparations.

Purification of DNA from very small amounts of starting material is

also possible. If the sample has less than 5 ng DNA (<10,000
genome copies), carrier DNA (a homopolymer such as poly dA,
poly dT, or gDNA) should be added to the starting material. It is
important to check that the carrier DNA does not interfere with your
downstream application.

Yields of DNA obtained using DNeasy Tissue technology are shown

in Table 3.

Genomic DNA Purification 7


Table 4. Factors influencing disruption efficiency
◆ Size and composition of beads
◆ Ratio of buffer to beads
◆ Amount of starting material
◆ Speed and configuration of agitator
◆ Disintegration time
8 Genomic DNA Purification
Disruption and lysis
Complete disruption and lysis of cell walls and plasma membranes
of cells and organelles is essential for all genomic DNA isolation
procedures. Incomplete disruption results in significantly reduced
yields. The methods listed below are suitable for the breakdown of
all cellular and organellar membranes. The DNA obtained will vary
in size between these methods due to differences in shearing applied
to the genomic DNA during lysis. It should be noted that some
protocols require prior isolation of cell nuclei, and will therefore yield
total nucleic genomic DNA, but not organelle-derived DNA (e.g.,
mitochondrial).
Disruption methods
Enzymatic lysis of animal tissues
Disruption generally involves the use of a lysis buffer that contains a
detergent (for breaking down cellular membranes) and a protease
(for digestion of protein cellular components). The choice of protease
depends on the lysis buffer used. Both QIAGEN Proteinase K and
QIAGEN Protease have high activity in buffers commonly used for
DNA isolation. QIAGEN Proteinase K is recommended for buffers
containing SDS and >8 mM EDTA.
Enzymatic lysis of bacteria and yeast
Additional enzymatic lysis should be used when isolating genomic
DNA from Gram-positive bacteria or yeast. The structure of the
Gram-negative bacterial cell wall means that additional enzymatic
lysis is unnecessary for these species. Lysozyme (for Gram-positive
bacteria) or lyticase (for yeast) should be added to the enzymatic
lysis buffer immediately before use.
Disruption using rotor-stator homogenizers
Rotor-stator homogenizers thoroughly disrupt animal tissues in
approximately 5–90 seconds depending on the toughness of the
sample. The rotor turns at very high speed disrupting the sample by
a combination of turbulence and mechanical shearing. Rotor-stator
homogenizers are available in different sizes and have probes of
different sizes. Probes with diameters of 5 mm and 7 mm are
suitable for volumes up to 300 µl and can be used in microcentrifuge
tubes. Probes with a diameter of 10 mm or greater require larger
tubes. This method can cause minor shearing of the genomic DNA
and may have a bearing on its molecular weight.
Disruption using the Mixer Mill MM 300 and other bead mills Efficient High-Throughput Disruption
of Tissue Samples
Disruption using a bead mill involves agitation at high speed in the
presence of beads. Disruption occurs by the shearing and crushing
action of the beads as they collide with the cells. Disruption efficiency
is influenced by the factors shown in Table 4 (page 8). The optimal
types of beads to use for disruption of various starting materials in
the Mixer Mill MM 300 are shown in Table 5.

Table 5. Starting material and optimal beads to use

Mean bead size

Starting sample Bead type (diameter)
Animal tissues Stainless steel 3–7 mm
Figure 3. The Mixer Mill MM 300 allows processing
Animal cells Glass 0.5 mm of up to 192 tissue samples in just 2–4 minutes.

Bacteria Glass 0.1 mm

Yeast Glass 0.5 mm

Glass beads should be pretreated before use by washing in

concentrated nitric acid. Alternatively, commercially available
acid-washed glass beads can be used. All other disruption
parameters should be determined empirically for each application.

Samples can be disrupted in either an appropriate buffer or in liquid

nitrogen. If disruption is performed in liquid nitrogen, it is critical to
ensure that the sample remains frozen at all times. Disruption using
liquid nitrogen in conjunction with a bead mill is comparable to that
achieved using a pestle and mortar (see below). If disruption is
performed in buffer, it is important to optimize the disruption time.
If disruption is prolonged once material has been lysed, significant
degradation can occur.

Disruption using a mortar and pestle

For disruption using a mortar and pestle, the sample is frozen
immediately in liquid nitrogen and ground to a fine powder using
liquid nitrogen. The suspension (tissue powder and liquid nitrogen) is
transferred into a liquid-nitrogen–cooled, appropriately sized tube
and the liquid nitrogen allowed to evaporate without thawing of the
sample. In order to minimize degradation, lysis buffer should be
added and the isolation procedure continued as quickly as possible.

Genomic DNA Purification 9

 DNA isolation methods
Many different methods and technologies are available for the
isolation of genomic DNA. In general, all methods involve disruption
and lysis of the starting material followed by the removal of proteins
and other contaminants and finally recovery of the DNA. Removal of
proteins is typically achieved by digestion with proteinase K,
followed by salting-out, organic extraction, or binding of the DNA to
a solid-phase support (either anion-exchange or silica technology).
DNA is usually recovered by precipitation using ethanol or
isopropanol. The choice of a method depends on many factors: the
required quantity and molecular weight of the DNA, the purity
required for downstream applications, and the time and expense.

Several of the most commonly used methods are detailed below,

although many different methods and variations on these methods
exist (a comparison of methods is shown in Figure 7, page 13).
Home-made methods often work well for researchers who have
developed and regularly use them. However, they usually lack
standardization and therefore yields and quality are not always
reproducible. Reproducibility is also affected when the method is
used by different researchers, or with different sample types.

The separation of DNA from cellular components can be divided into

four stages:

1. Disruption
2. Lysis
3. Removal of proteins and contaminants
4. Recovery of DNA

In some methods, stages 1 and 2 are combined.

Preparation of crude lysates

An easy technique for isolation of genomic DNA is to incubate cell
lysates at high temperatures (e.g., 90°C for 20 minutes), or to
perform a proteinase K digestion, and then use the lysates directly in
downstream applications. Considered ”quick-and-dirty” techniques,
these methods are only appropriate for a limited range of
applications. The treated lysate usually contains enzyme-inhibiting
contaminants, such as salts, and DNA is often not at optimal pH (1).
Furthermore, incomplete inactivation of proteinase K can result in
false negative results and high failure rates. It is not recommended to
store DNA prepared using this method, as the high levels of
contamination often result in DNA degradation.

10 Genomic DNA Purification

Salting-out methods
Starting with a crude lysate, ”salting-out” is another conventional
technique where proteins and other contaminants are precipitated
from the cell lysate using high concentrations of salt such as
potassium acetate or ammonium acetate (2). The precipitates are
removed by centrifugation, and the DNA is recovered by alcohol
precipitation. Removal of proteins and other contaminants using this
method may be inefficient, and RNase treatment, dialysis, and/or
repeated alcohol precipitation are often necessary before the DNA
can be used in downstream applications. DNA yield and purity are
highly variable using this method.

Organic extraction methods

Organic extraction is a conventional technique that uses organic
solvents to extract contaminants from cell lysates (3, 4). The cells are
lysed using a detergent, and then mixed with phenol, chloroform,
and isoamyl alcohol. The correct salt concentration and pH must be
used during extraction to ensure that contaminants are separated into
the organic phase and that DNA remains in the aqueous phase.
DNA is usually recovered from the aqueous phase by alcohol
precipitation. This is a time-consuming and cumbersome technique.
Furthermore, the procedure uses toxic compounds and may not give
reproducible yields (5). DNA isolated using this method may contain
residual phenol and/or chloroform, which can inhibit enzyme reactions
in downstream applications, and therefore may not be sufficiently
pure for sensitive downstream applications such as PCR (6). The
process also generates toxic waste that must be disposed of with
care and in accordance with hazardous waste guidelines. In addition,
this technique is almost impossible to automate, making it unsuitable
for high-throughput applications.

Cesium chloride density gradients

Genomic DNA can be purified by centrifugation through a cesium
chloride (CsCl) density gradient. Cells are lysed using a detergent,
and the lysate is alcohol precipitated. Resuspended DNA is mixed
with CsCl and ethidium bromide and centrifuged for several hours.
The DNA band is collected from the centrifuge tube, extracted with
isopropanol to remove the ethidium bromide, and then precipitated
with ethanol to recover the DNA. This method allows the isolation of
high-quality DNA, but is time consuming, labor intensive, and
expensive (an ultracentrifuge is required), making it inappropriate for
routine use. This method uses toxic chemicals and is also impossible
to automate.

Genomic DNA Purification 11

 DNeasy Tissue Anion-exchange methods
Spin and 96-Well Plate Procedures
Solid-phase anion-exchange chromatography is based on the
Tissue sample Mouse tail or animal tissue interaction between the negatively charged phosphates of the nucleic
acid and positively charged surface molecules on the substrate. DNA
binds to the substrate under low-salt conditions, impurities such as
Lyse Collect RNA, cellular proteins, and metabolites are washed away using
mouse tails or
tissue samples medium-salt buffers, and high-quality DNA is eluted using a high-salt
and lyse buffer. The eluted DNA is recovered by alcohol precipitation, and is
suitable for all downstream applications.

Anion-exchange technology completely avoids the use of toxic

Bind DNA Bind DNA substances, and can be used for different throughput requirements as
well as for different scales of purification. The isolated DNA is sized
up to 150 kb, with an average length of 50–100 kb. QIAGEN
offers QIAGEN Genomic-tips for the purification of high-molecular-
weight DNA.

Silica-based methods — DNeasy Tissue Kits

Wash Wash DNeasy Tissue technology provides a simple, reliable, fast, and
inexpensive method for isolation of high-quality DNA. This method is
based on the selective adsorption of nucleic acids to a silica-gel
membrane in the presence of high concentrations of chaotropic salts
(Figure 4). Use of optimized buffers in the lysis procedure ensures
that only DNA is adsorbed while cellular proteins, and metabolites
Elute
remain in solution and are subsequently washed away. This is sim-
pler and more effective than other methods where precipitation or
extraction is required. Ready-to-use DNA is then eluted from the
Ready-to-use DNA
silica-gel membrane using a low-salt buffer. No alcohol precipitation
Elute into Elution
is required, and resuspension of the DNA, which is often difficult if
Microtubes RS the DNA has been over-dried, is not required.
Ready-to-use DNA
DNeasy Tissue Kits are designed for rapid isolation of pure total
DNA (genomic, viral, and mitochondrial) from a wide variety of
Figure 4. The DNeasy Tissue spin and 96-well plate sample sources, including fresh and frozen animal cells and tissues,
procedures.
yeasts, and blood. DNA purified using DNeasy Tissue Kits is free
from contamination and enzyme inhibitors and is highly suited for
applications such as Southern blotting, PCR, real-time PCR, RAPD,
RFLP, and AFLP analyses. DNeasy Tissue Kits are available in
convenient spin-column or 96-well formats, suitable for a wide range
of throughput needs.

Genomic DNA isolated using DNeasy Tissue technology is up to

50 kb in size, with an average length of 20–30 kb. DNA of this
length is particularly suitable for PCR analysis as well as Southern
blotting analysis (7–13). Silica-gel spin technology is not suitable if
genomic DNA >50 kb is required for certain cloning or blotting
applications. QIAGEN recommends the use of QIAGEN Genomic-tips
for these applications.

12 Genomic DNA Purification

The DNeasy Tissue procedure is suitable for both very small and Effect of Sample Size on DNA Yield
large sample sizes, from as little as 100 cells up to 5 x 106 cells. In
order to obtain optimal DNA yield and quality, it is important not to 50 Liver
Salivary gland
overload the DNeasy System, as this can lead to significantly lower Kidney
yields than expected (Figure 5). Overloading the DNeasy System can 40
also adversely affect the purity of the DNA (Figure 6).

DNA yield (µg)

The DNeasy Tissue procedure is also highly suited for purification of 30
DNA from very small amounts of starting material. If the sample has
less than 5 ng DNA (<10,000 copies), 3–5 ng carrier DNA 20
(a homopolymer such as poly dA, poly dT, or gDNA) should be
added to the starting material. Ensure that the carrier DNA does not
10
interfere with the downstream application.

0
10 15 20 25 30
Tissue (mg)

DNA Purity and Time Required for Different Isolation Methods

Figure 5. DNA was purified from tissue samples
pooled from 10 mice using the DNeasy Tissue protocol
High for rodent tails. DNA yield was determined spectro-
QIAGEN QIAGEN photometrically. Each point represents the mean and
Anion- 1 x CsCl standard deviation from 10 preparations.
DNeasy gradient
exchange
Tissue Kits
technology

Organic Effect of Sample Size on DNA Purity

Purity

extraction
2.1
Salting- Liver
alcohol out Salivary gland
Alcohol Kidney
precipi-
precipitation
Simple tation 2.0
methods*
A260/A280 ratio

Low
1.9
Less Time More

Figure 7. Length of time taken for the common methods of genomic DNA isolation, 1.8
after proteinase K digestion. Simple methods are defined as boiling preps and other
protocols that do not include a lysis step. Alcohol precipitation is the precipitation of
proteinase K-digested lysates after removal of insoluble particles by centrifugation. For
organic extraction phenol/chloroform is added to the proteinase K-digested lysate, 1.7
vortexed and centrifuged. The upper phase is then subject to alcohol precipitation. 10 15 20 25 30
Salting-out is defined as treatment of the proteinase K-digested lysate with a high-salt
buffer. This is incubated and the proteins precipitated by centrifugation. The supernatant Tissue (mg)
is then subject to alcohol precipitation. See pages 10–12 for further information on DNA
isolation methods.
Figure 6. The DNA purity of the samples described in
* No proteinase K digestion.
Figure 5 was determined spectrophotometrically by
measuring the A260/A280 ratio.

Genomic DNA Purification 13

 Applications using DNeasy Tissue Kits
DNeasy Tissue and DNeasy 96 Tissue Kits provide simple, rapid,
and reproducible methods for the isolation of DNA from a wide
range of sample sources. DNA purified using DNeasy 96 Tissue is
suitable for a broad range of downstream applications such as PCR,
Southern blotting, RFLP, AFLP, SNP analysis, microsatellite analysis,
and Masscode technology.

Some examples of the uses of DNeasy Tissue Kits are given in the
following pages. This section contains many applications currently
performed using DNeasy Tissue Kits. It provides a guide to the range
of uses of DNeasy Tissue technology.

Functional genomics
Functional genomics is used to determine the biological function of
nucleic acid sequences. A wide range of techniques are used
including screening of transgenic and knockout animals (usually
mice) and mutant analysis. DNeasy Tissue Kits provide a rapid and
easy method for the isolation of genomic DNA for such analyses.

Identification of Nonfunctional Genes by PCR

+/+ –/–
o1

o2

o1

o2
ne

ne

ne

ne
t

t
w

w
1018

506

396

Figure 8. Mice with a nonfunctional CRALBP (cellular retinaldehyde binding protein)

gene (Rlbp1-/Rlbp1- mice) were generated. In humans, mutations in the CRALBP gene
cause retinal pathology and delayed dark adaptation. The primary biochemical role of
this gene is unknown, and knockout mice were generated to address this question. The
DNeasy Tissue Kit was used to purify DNA from tail tips taken from the offspring of mice
chimeric for a nonfunctional version of the CRALBP gene. Primers were designed to
amplify either wild-type (wt) or targeted (neo1 and neo2) alleles, and used to amplify
DNA from mice of interest. This method yielded high-quality DNA that gave reliable
results in genotyping by PCR.

Excerpted from Saari, J.C., Nawrot, M., Kennedy, B.N., Garwin, G.G., Hurley, J.B., Huang, J., Possin, D.E., and
Crabbs, J. W. (2001) Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results
in delayed dark adaptation. Neuron 29, 739. Published with permission.

14 Genomic DNA Purification

 Genotyping of Knockout Animals Using DNeasy Tissue Technology

1 2 3

– 500 bp
– 300 bp

Figure 9. The role of the Bax protein in nerve cell death, and the response of central
nervous system cells to oxidative stress was examined in knockout mice. Bax-
homozygous knockout mice were bred by crossing Bax-heterozygous mice, so that a
colony and a source of embryos lacking the Bax protein were maintained. Genomic
DNA was isolated from pups using the DNeasy Tissue Kit and the genotype of individual
mice was determined by PCR. Bax is not expressed in homozygous Bax-deleted animals.
Lane 1: Bax –/+, Lane 2: Bax +/+, Lane 3: Bax –/–.

Data excerpted from Dargusch, R., Piasecki, D., Tan, S., Liu, Y., and Schubert, D. (2001) The role of Bax in glutamate-
induced nerve cell death. Journal of Neurochemistry 76, 295. Published with permission.

Genotyping of Transgenic Tadpoles by PCR

co l
l
FP tro

ro
nt
G on
c
oc

– + + + + + + M
N

– 500 bp
GFP →
Noc → – 250 bp

1 2 3 4 5 6 7

Figure 10. Transgenic Xenopus were used to investigate the regulatory mechanism of
nocturnin, the vertebrate circadian clock-regulated gene in Xenopus laevis. The in vivo
expression patterns of GFP reporters driven by various portions of the nocturnin gene
were analyzed. To identify transgenic tadpoles, genomic DNA was isolated from the
clipped tails of tadpoles using the DNeasy Tissue Kit, and analyzed by PCR. Endogenous
nocturnin was amplified to monitor the quality of the genomic DNA, and the GFP coding
sequence was amplified to examine whether the GFP constructs had integrated into the
tadpole genome. The data shows the results from various transgenic tadpoles
(tadpoles 2–7), and a nontransgenic tadpole (tadpole 1). Endogenous nocturnin bands
were seen in all of the tadpoles whereas the GFP bands were observed only in the
transgenic tadpoles.

Data excerpted from Liu, X., and Green, C.B. (2001) A novel promoter element, photoreceptor conserved element II,
directs photoreceptor-specific expression of nocturnin in Xenopus laevis. J. Biol. Chem. 276, 15146. Published with
permission.

Genomic DNA Purification 15

 Molecular breeding
Molecular markers provide a powerful tool for understanding the
genetic basis of traits, especially those that involve several loci.
Animal breeders use molecular markers to select for, and breed
animals with improved characteristics such as improved growth, and
disease resistance. This technology can also be used to maintain or
improve the genetic variation in farmed species.

Microsatellite Analysis of Farmed Atlantic Salmon

FAM -51 TAMRA -92

JOE/S -36 JOE/S -42 TAMRA -31 FAM27 JOE/1–8

¢ parent
Allele size
ranges

— JOE/L

— TAMRA

— FAM
™ parent

Offspring
— JOE/S

Figure 11. Genetic profiling of farmed Atlantic salmon (Salmo salar) was performed in
conjunction with a breeding program aimed at improving performance of stocks,
maintaining and maximizing genetic variation, and determining parentage, thus
preventing inbreeding. Total DNA was isolated from 5–10 mg Atlantic salmon fin tissue
stored in ethanol, using the standard "DNeasy 96 Tissue Protocol for High-Throughput
DNA Isolation from Rodent Tails and Animal Tissues” with the following modifications.
An increased volume of proteinase K was used for digestion of residual tissue in step 2
of the protocol. In step 15, plates were centifuged for 15 minutes to ensure complete
ethanol removal. DNA was eluted using 200 µl elution buffer and 1 µl was used for mul-
tiplex PCR of four microsatellite loci.

Data kindly provided by Dr. H. Sobolewska and Dr. A. Hamilton, Landcatch Natural Selection, The E-Centre,
Cooperage Way Business Park, Alloa, Clackmannanshire FK10 3LP UK.

16 Genomic DNA Purification

 Genetic studies in bacteria and yeast
The study of bacteria is a diverse field, with far-reaching implications
for human health and disease, animal husbandry, agriculture and
aquaculture, the environment, and the food and brewing industries.
Studies at the genetic level in bacteria and yeast have led to
significant advances in these areas.

RFLP Analysis of Borrelia burgdorferi DNA

CC

kb

01

02

03
87

88

89

90

91

92

93

94

95

96

97

98

99

00
13
3
5A

15

15

15
14

14

14

14

14

14

14

14

14

14

14

14

14

15
B3
AT

9.0
8.0 –
7.0
6.0 –
5.0 –
4.0 –

3.0 –

2.0 –
1.6 –

1.0 –

0.5 –

Figure 12. Total bacterial DNA was isolated from B. burgdorferi, the causative agent of
Lyme disease, using the DNeasy Tissue Kit. Possible variations in bdr-flanking regions
were assessed using an RFLP-based approach. RFLP analysis indicating stability of
bdr-flanking regions. Southern blots of total B. burgdorferi DNA digested with XbaI were
probed with a pool of PCR-derived bdr probes. Three in vitro cultured B31 clones
(5A3, ATCC, and B313) and 17 isolates obtained from mice 1 year post-infection with
B31-5A3 are shown. This study demonstrated that apart from plasmid loss during in
vitro cultivation, the bdr paralog loci of strain B31 are stable. This suggests that
recombinatorial variation of bdr genes is not essential for persistent mammalian
infection. These loci could provide new tools for plasmid profiling and strain typing, and
could be especially useful for quickly assessing possible variations of complex Borrelia
populations present in vector ticks or mouse reservoirs.

Data excerpted from Zückert, W.R. and Barbour, A.G. (2000) Stability of Borrelia burgdorferi bdr loci in vitro and in
vivo. Infection and Immunity 68, 1727. Published with permission.

Genomic DNA Purification 17

 Disease research
Studies in human or animal cell lines and the use of animal models
has led to a greater understanding of human diseases and their
treatment. DNeasy Tissue technology is highly suited for the
purification of high-quality genomic DNA from a wide range of cell
lines and animal tissues.

Undifferentiated FLEC in Post-Transplantation Livers

A
DN
Without PH With PH Normal ¢

l™

A
DN
ro

5
05

00
nt
5

5
M

o
Co
50

50

50
ng DNA

0.

0.

0.

0.

0.

N
5

5
— 200 bp
— 100 bp

Figure 13. Adult rats were treated with retrorsine to prevent hepatocyte proliferation, and
then, in conjunction with a partial hepatectomy (PH) fetal liver epithelial cells (FLEC) were
transplanted into their livers. FLEC were also transplanted into the livers of untreated rats.
The DNeasy Tissue Kit was used to isolate DNA from rat livers and PCR analysis of the
rat sry gene, located on the Y chromosome in transplanted cells, determined whether
undifferentiated FLEC remained after transplantation. It was demonstrated that large
numbers of undifferentiated FLEC (and their progeny) were present in livers that had
undergone a partial hepatectomy, while only small numbers remained in livers that were
functioning normally (i.e., no partial hepatectomy). M: markers.

Data excerpted from Dabeva M.D., Petkov, P.M., Sandhu, J., Oren, R., Laconi, E., Hurston, E., and Shafritz, D.A.
(2000) Proliferation and differentiation of fetal liver epithelial progenitor cells after transplantation into adult liver.
Am. J. Path. 156, 2017. Published with permission.

Real-Time PCR Detection of a Tumor-Specific Chromosomal Translocation

A B

Copies of tumor-specific translocation

7
70
700
7000

Figure 14. DNA was purified using the DNeasy Tissue Kit. A chromosomal translocation in the Burkitt’s lymphoma cell line
Ramos was detected using real-time PCR. Various numbers (7, 70, 700, and 7000 copies) of the tumor-specific translocation
were spiked into 250 ng translocation-free human genomic DNA (also purified using the DNeasy Tissue Kit). All quantities
were reproducibly detected in real-time PCR using the QIAGEN QuantiTect ™ Probe PCR Kit and the “Protocol for Quantitative,
Real-Time PCR Using ABI Sequence Detection Systems and Other Real-Time Thermal Cyclers”. Analysis was performed on the
ABI PRISM® 7700 Sequence Detection System. Dual-labeled probes for the 5'–3' nuclease assay contained either A: 6-FAM
as the reporter and Black Hole Quencher™1 (translocation-specific probe) or B: TAMRA as the reporter and Black Hole
Quencher 2 (GAPDH-specific probe). The coefficient of determination (R 2) for A was 0.9997.

18 Genomic DNA Purification

Natural history applications
Old dried skins, skeletons, and formalin-fixed, ethanol-preserved
animal specimens represent a potentially valuable source of data,
especially where the animal in question is rare or extinct. However,
the difficulties of obtaining usable DNA from preserved specimens
are many. Bones have been found to be a much more reliable source
of DNA, especially of long DNA fragments.

Successful Amplification of DNA

from 8- and 19-Year-Old Bat Specimens

Short digestion Long digestion

Washed Unwashed Washed Unwashed

Dry bone A

Wet bone B

Wet skin C

Controls D

Figure 15. Small bones (a few millimeters long) and skin from fruit bats of the genus
Sturnira were used. The DNeasy Tissue protocol was modified to include additional
washing and rehydration of tissues, prolonged proteinase K digestion, adjustment of pH
before adsorption of DNA onto the DNeasy spin column, and reduction in the elution
volume. This provided amplifiable cytochrome b DNA suitable for PCR. The data shows
amplification of cytochrome b from an 8-year-old bat museum specimen preserved in
formalin/ethanol (wet material) and from a 19-year-old bat specimen (dry material).
Each sample was amplified using a forward primer (cyb 8) and one of four reverse
primers, each yielding a product of successively greater length. Reverse primers and
approximate length of product when combined with forward primer cyb 8 were: 1R
(279 bp); 2R (494 bp); 3R (698 bp); and cyb 7 (879 bp). Variables examined were tis-
sue source, duration of rehydration (washed vs. unwashed), and duration of digestion
(3 h vs. 72 h). The best amplifications and PCR products were obtained from dry bones
digested for 72 h. Washing and rehydration before extraction appeared to have no pos-
itive effect. Amplification from skin samples was inferior to those obtained from bone.
Results from dry skin are not shown but were similar to wet skin. Positive control template
was bat DNA from saturated EDTA-DMSO saline (SED) buffer-preserved fresh kidney.

Data excerpted from Iudica, C.A., Whitten, W.M., and Williams, N.H. (2001) Small bones from dried mammal
museum specimens as a reliable source of DNA. BioTechniques 30 734. Published with permission.

Genomic DNA Purification 19

 Phylogeny and biodiversity studies
Phylogenetic studies are essential for understanding evolutionary
history and species diversity. Furthermore, where species are
endangered, phylogenetic analysis provides a valuable insight to the
conservation risks to these species. Mouse lemurs are the world’s
smallest living primates and are native to Madagascar. Detailed
investigation of species diversity for mouse lemurs (genus
Microcebus) is essential for evaluating the above factors to these
organisms.

Madagascan Mouse Lemur Phylogenetic Diversity in Madagascan Mouse Lemurs

100
M. ravelobensis

100
M. tavaratra

99 M. myoxinus
59 66
85
100 M. berthae

99
63
Figure 16. The mouse lemur, which is native to “M. rufus1”
Madagascar. (Photograph courtesy of David Haring, 100
98
Duke University Primate Center, Durham, NC, USA).
100 “M. rufus2”
98
M. sambiranensis

M. murinus

100
M. griseorufus

Figure 17. Genomic DNA from liver, spleen, kidney, or ear punches from individuals
from an extensive array of localities was isolated using DNeasy Tissue Kits and was used
to examine the species diversity of Microcebus at the genetic level. Several mitochondrial
DNA markers (mtDNA) were selected in order to show genetic variation in the inter- and
intraspecific level. The figure shows the phylogeny derived from sequence alignment of
2404 bp of combined mtDNA sequences from the control region homologous with the
hypervariable region 1 in humans, COII and cytochrome b. Clades are color-coded to
emphasize species diversity. The analysis showed unexpectedly high levels of species
diversity among mouse lemurs, which was previously underestimated.

Data excerpted from Yoder, A.D., Rasoloarison, R.M., Goodman, S.M., Irwin, J.A., Atsalis, S., Ravosa, M.J., and
Ganzhorn, J.U. (2000) Remarkable species diversity in Malagasy mouse lemurs (primates Microcebus) Proc. Natl.
Acad. Sci. USA. 97, 11325. Published with permission.

20 Genomic DNA Purification

Detection of viral pathogens
Animal viruses can cost farmers and animal breeders large sums of
money per year in treatment and lost animals. The DNeasy Tissue Kit
provides an efficient method for the isolation of viral DNA.

PCR and Nested PCR to Detect PCV2 in Boar Semen and Serum

Serum Semen Controls

en
m
se
nP PC trol
R: dpi

ne 47 i

PC ve i
p
dp

on
i
R: dpi

V2
dp

d
R: dp

dp

dp

C V2
R: i

R: i
dp

dp

c
nP 28

nP 21

PC
nP 28
PC 0

PC 4

nP 0

nP : 5
0

ti
R:

R:

R:

R:
R

ga
R:

R:
C

C
M M
PC

nP

nP

– 894 bp

– 263 bp

Figure 18. The presence of porcine circovirus type 2 (PCV2) in the semen of infected
boars was investigated. Four boars were infected with PCV2 and 2 boars were
inoculated with non-infected cells as controls. The DNeasy Tissue Kit was used to isolate
DNA from serum and semen samples for several days post-infection (dpi), and PCV2
nucleic acid was detected using PCR and nested PCR. Following infection, PCV2 DNA
was detected in semen concurrently with the presence of PCV2 DNA and antibodies in
serum, and PCV2 was shed intermittently in the semen of infected boars. Controls from
left to right: negative control semen, PCR of PCV2 isolate diluted in uninfected semen,
nested PCR of PCV2 isolate diluted in uninfected semen. PCR: PCR, nPCR: nested PCR,
M: markers.

Data excerpted from Larochelle, R., Bielanski, A., Müller, P., and Magar, R. (2000) PCR detection and evidence of
shedding in porcine circovirus type 2 in boar semen. J. Clin. Microbiol. 38, 4629. Published with permission.

Genomic DNA Purification 21

 Protocols
The standard DNeasy Tissue protocols for purification of DNA from
rodent tails, animal tissues, and cultured cells, are detailed here (see
also the DNeasy Procedure flowchart on page 12). DNeasy Tissue
technology is very versatile and can also be successfully used to
isolate genomic DNA from bacteria, yeast, animal blood, insects,
and fixed tissues. These protocols are given below. Furthermore,
although the protocols detailed here refer only to DNeasy Tissue spin
column technology, DNeasy Tissue technology is also available in a
convenient high-throughput (96-well plate) format.

In addition, protocols are listed for the isolation of genomic DNA

from saliva, nails, hair, or bird feathers, and from compact bone.
These are User-Developed Protocols, developed by customers for their
own applications. QIAGEN is continually developing and optimizing
DNeasy Tissue protocols for new sample sources, which are not
listed here.

Current standard and User-Developed protocols are always available

from QIAGEN Technical Service. Please contact QIAGEN Technical
Services or your local distributor to receive free copies of any of
these protocols or to inquire about other applications.

DNeasy Tissue and DNeasy 96 Tissue Handbooks contain all the

standard DNeasy protocols, and all standard and a selection of
User-Developed Protocols are available to download, view, and
print at www.qiagen.com/literature/protocols.

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.
Please contact us if you have any suggestions about product
performance or new applications and techniques.

22 Genomic DNA Purification

DNeasy protocol for animal tissues
1. Cut up 25 mg tissue (or up to 10 mg spleen)
into small pieces, place in a 1.5 ml micro-
centrifuge tube, and add 180 µl Buffer ATL.
It is advisable to cut the tissue into small pieces
for efficient lysis.
2. Add 20 µl proteinase K, mix by vortexing, and
incubate at 55°C until the tissue is completely
lysed.
Vortex occasionally during incubation to
disperse the sample, or place in a shaking
water bath on a rocking platform.
Lysis time varies depending on the type of tissue
being processed. Lysis is usually complete in
1–3 h, though samples can be lysed overnight.
Optional: RNase treatment of the sample. Add
4 µl RNase A (100 mg/ml), mix by vortexing,
and incubate for 2 min at room temperature.
Transcriptionally active tissues such as liver and
kidney contain high levels of RNA, which will
copurify with genomic DNA. If RNA-free
genomic DNA is required, carry out this step.
3. Vortex for 15 s. Add 200 µl Buffer AL to the
sample, mix thoroughly by vortexing, and
incubate at 70°C for 10 min.
4. Add 200 µl ethanol (98–100%) to the sample,
and mix thoroughly by vortexing.
A white precipitate may form on addition of
ethanol. It is essential to apply all of the
precipitate to the DNeasy spin column.
5. Pipet the mixture from step 4 into the DNeasy
spin column placed in a 2 ml collection tube
(provided). Centrifuge at ≥6000 x g (8000 rpm)
for 1 min. Discard flow-through and collection
tube.
6. Place the DNeasy spin column in a new 2 ml
collection tube (provided), add 500 µl
Buffer AW1, and centrifuge at ≥6000 x g
(8000 rpm) for 1 min. Discard flow-through
and collection tube.
7. Place the DNeasy spin column in a new 2 ml
collection tube (provided), add 500 µl
Buffer AW2, and centrifuge for 3 min at full
Genomic DNA Purification
speed to dry the DNeasy membrane. Discard
flow-through and collection tube.
This step ensures that no residual ethanol is
carried over during the following elution.
Remove the spin column carefully to ensure that
the column does not touch the flow-through.
8. Place the DNeasy spin column in a clean
1.5 ml or 2 ml collection tube (not provided),
and pipet 200 µl Buffer AE directly onto the
DNeasy membrane. Incubate at room
temperature for 1 min, and then centrifuge for
1 min at ≥6000 x g (8000 rpm) to elute.
9. Repeat elution once as described in step 8.
Elution can be performed in the same or
separate tubes. Do not use more than 200 µl
Buffer AE as this will cause the eluate to come
into contact with the DNeasy spin column.
DNeasy protocol for rodent tails
1. Cut one (rat) or up to two (mouse) 0.4–0.6 cm
lengths of tail into a 1.5 ml microcentrifuge
tube. Add 180 µl Buffer ATL. Earmark the
animal appropriately.
A maximum of 1.2 cm (mouse) or 0.6 cm (rat)
tail should be used. When purifying the DNA
from the tail of an adult mouse or rat, it is
recommended to use only 0.4–0.6 cm.
2. Add 20 µl proteinase K, mix by vortexing, and
incubate at 55°C until the tissue is completely
lysed. Vortex occasionally during incubation to
disperse the sample, or place the tube in a
shaking water bath on a rocking platform.
After mixing the tail section with proteinase K,
ensure the tail section is fully submerged. Lysis
is usually complete in 6–8 h, but samples can
be lysed overnight.
Optional: RNase treatment of the sample. Add
4 µl RNase A (100 mg/ml), mix by vortexing,
and incubate for 2 min at room temperature.
Rodent tail contains low levels of RNA, which
will be copurified. RNase digestion can be
used to destroy any residual RNA.
23
 3. Vortex for 15 s. Add 400 µl Buffer AL–ethanol
mixture to the sample, and mix thoroughly by
vortexing.
A white precipitate may form on addition of
ethanol. It is essential to apply all of the
precipitate to the DNeasy spin column.
4. Pipet the mixture from step 3 into the DNeasy
spin column placed in a new 2 ml collection
tube (provided). Centrifuge at ≥6000 x g
(8000 rpm) for 1 min. Discard flow-through
and collection tube.
5. Place the DNeasy spin column in a new 2 ml
collection tube (provided), add 500 µl
Buffer AW1, and centrifuge at ≥6000 x g
(8000 rpm) for 1 min. Discard flow-through
and collection tube.
6. Place the DNeasy spin column in a new 2 ml
collection tube (provided), add 500 µl
Buffer AW2, and centrifuge for 3 min at full
speed to dry the DNeasy membrane. Discard
flow-through and collection tube.
This step ensures that no residual ethanol is
carried over during the following elution.
Remove the spin column carefully to ensure that
the column does not touch the flow-through.
7. Place the DNeasy spin column in a clean
1.5 ml or 2 ml collection tube (not provided),
and pipet 200 µl Buffer AE directly onto the
DNeasy membrane. Incubate at room
temperature for 1 min, and then centrifuge for
1 min at ≥6000 x g (8000 rpm) to elute.
8. Repeat elution once as described in step 7.
Elution can be performed in the same or
separate tubes. Do not use more than 200 µl
Buffer AE as this will cause the eluate to come
into contact with the DNeasy spin column.
DNeasy protocol for cultured animal cells
1. Centrifuge the appropriate number of cells
(max. 5 x 106) for 5 min at 300 x g.
Resuspend pellet in phosphate buffered saline
(PBS, not supplied).
24
When using a frozen cell pellet, before adding
PBS allow cells to thaw until the pellet can be
dislodged by gently flicking the tube.
Optional: If RNA-free genomic DNA is
required, add 4 µl RNase A (100 mg/ml) and
incubate for 2 min at room temperature.
2. Add 20 µl proteinase K and 200 µl Buffer AL to
the sample, mix thoroughly by vortexing, and
incubate at 70°C for 10 min. Do not add
proteinase K directly to Buffer AL.
3. Add 200 µl ethanol (96–100%) to the sample
and mix thoroughly by vortexing. A white
precipitate may form on addition of ethanol. It
is essential to apply all of the precipitate to the
DNeasy spin column.
4. Pipet the mixture from step 3 into the DNeasy
spin column placed in a new 2 ml collection
tube (provided). Centrifuge at ≥6000 x g
(8000 rpm) for 1 min. Discard flow-through
and collection tube.
5. Place the DNeasy spin column in a new 2 ml
collection tube (provided), add 500 µl Buffer
AW1, and centrifuge at ≥6000 x g (8000 rpm)
for 1 min. Discard flow-through and collection
tube.
6. Place the DNeasy spin column in a 2 ml
collection tube (provided), add 500 µl
Buffer AW2, and centrifuge for 3 min at full
speed to dry the DNeasy membrane. Discard
flow-through and collection tube.
This step ensures that no residual ethanol is
carried over during the following elution.
Remove the spin column carefully to ensure that
the column does not touch the flow-through.
7. Place the DNeasy spin column in a clean
1.5 ml or 2 ml collection tube (not provided),
and pipet 200 µl Buffer AE directly onto the
DNeasy membrane. Incubate at room
temperature for 1 min, and then centrifuge for
1 min at ≥6000 x g (8000 rpm) to elute.
8. Repeat elution once as described in step 7.
Elution can be performed in the same or
separate tubes. Do not use more than 200 µl
Buffer AE as this will cause the eluate to come
into contact with the DNeasy spin column.
Genomic DNA Purification
Preparation and lysis of other starting materials
The protocols detailed below generally only differ
from the standard protocols (listed above) at the
sample preparation stage.
Protocols for animal blood
These protocols can be used for the isolation of
genomic DNA from animal blood, as well as buffy
coat and bone marrow.
Protocol for whole non-nucleated blood
For use with mouse, rat, guinea pig, hamster, rabbit,
cow, and monkey blood.
1. Pipet 20 µl proteinase K into the bottom of a
1.5 ml microcentrifuge tube (not provided).
2. Add 50–100 µl anticoagulated blood.
3. Adjust the volume to 220 µl with PBS.
4. Add 200 µl Buffer AL. Mix thoroughly by
vortexing.
5. Incubate for 10 min at 70°C.
6. Continue with step 3 of the “DNeasy Protocol
for Cultured Animal Cells”.
Protocol for whole nucleated blood
For use with chicken and goldfish blood.
1. Pipet 20 µl proteinase K into the bottom of a
1.5 ml microcentrifuge tube (not provided).
2. Add 5–10 µl anticoagulated blood.
3. Adjust the volume to 220 µl with PBS.
4. Add 200 µl Buffer AL. Mix thoroughly by
vortexing.
5. Incubate for 10 min at 70°C.
6. Continue with step 3 of the “DNeasy Protocol
for Cultured Animal Cells”.
Genomic DNA Purification
Protocols for fixed tissues
The DNeasy Tissue Kit can be used to isolate
genomic DNA from fixed tissues. The length of DNA
isolated depends on the age and type of sample, as
well as the method of fixative used, but is usually
<650 bp. Use of fixatives such as alcohol and
formalin is recommended. Fixatives that cause
cross-linking, such as osmic acid, are not
recommended as it can be difficult to obtain
amplifiable DNA from tissues fixed with these
reagents.
Lysis times will vary depending on the sample and
type of tissue.
Yields will depend on size and age of the sample.
Yields lower than those obtained using fresh or
frozen tissues are to be expected. Therefore eluting
in 50–100 µl Buffer AE is recommended.
Protocol for paraffin-embedded tissue
1. Place a small section (not more than 25 mg)
of paraffin-embedded tissue in a 2 ml
microcentrifuge tube.
2. Add 1200 µl xylene. Vortex vigorously.
3. Centrifuge at full speed for 5 min at room
temperature.
4. Remove supernatant by pipetting. Do not
remove any of the pellet.
5. Add 1200 µl absolute ethanol to the pellet to
remove residual xylene and mix gently by
vortexing.
6. Centrifuge at full speed for 5 min at room
temperature.
7. Carefully remove the ethanol by pipetting. Do
not remove any of the pellet.
8. Repeat steps 5–7 once.
9. Incubate the open microcentrifuge tube at 37°C
for 10–15 min until the ethanol has evaporated.
10. Resuspend the tissue pellet in 180 µl Buffer ATL
and continue with the “DNeasy Protocol for
Animal Tissues” from step 2.
25
 Protocol for formalin-fixed tissue
1. Wash tissue sample twice with PBS to remove
fixative.
2. Discard PBS and continue with the “DNeasy
Protocol for Animal Tissues”.
Protocols for bacteria
Protocol for Gram-negative bacteria
1. Harvest cells (max. 2 x 109 cells) in a micro-
centrifuge tube by centrifuging for 10 min at
7500 rpm (5000 x g). Discard supernatant.
2. Resuspend pellet in 180 µl Buffer ATL.
3. Continue with the ”DNeasy Protocol for Animal
Tissues” from step 2.
Protocol for yeasts
1. Harvest cells (max. 5 x 107 cells) in a micro-
centrifuge tube by centrifuging for 10 min at
7500 rpm (5000 x g). Discard supernatant.
2. Resuspend the pellet in 600 µl sorbitol buffer
(1 M sorbitol; 100 mM EDTA; 14 mM
β-mercaptoethanol). Add 200 units lyticase and
incubate at 30°C for 30 min. Lysis time and
yield will vary depending on cell number and
yeast species.
3. Pellet the spheroplasts by centrifuging for
10 min at 300 x g.
4. Resuspend the spheroplasts in 180 µl Buffer
ATL.
5. Continue with the “DNeasy Protocol for Animal
Tissues” from step 2.

Protocol for Gram-positive bacteria

1. Harvest cells (max. 2 x 109 cells) in a micro- Protocols for insects
centrifuge tube. By centrifuging for 10 min at Two protocols exist for the isolation of genomic
7500 rpm (5000 x g). Discard supernatant. DNA from insects, either can be used as desired.

2. Resuspend pellet in enzymatic lysis buffer (not

Protocol A
provided; 20 mM Tris·Cl, pH 8.0; 2 mM EDTA;
1.2% Triton® X-100; 20 mg/ml lysozyme). Add 1. Grind up to 50 mg insects in liquid nitrogen
lysozyme to buffer immediately before use. with a mortar and pestle, and place powder in
a 1.5 ml microcentrifuge tube.
3. Incubate for at least 30 min at 37°C.
2. Add 180 µl Buffer ATL.
4. Add 25 µl proteinase K and 200 µl Buffer AL.
Mix by vortexing. 3. Continue with the “DNeasy Protocol for Animal
Do not add proteinase K directly to Buffer AL. Tissues” from step 2.

5. Incubate at 70°C for 30 min. If required,

Protocol B
incubate at 95°C for 15 min to inactivate
pathogens. Note that incubation at 95°C 1. Place up to 50 mg insects in a 1.5 ml micro-
can lead to some DNA degradation. centrifuge tube.

6. Continue with the “DNeasy Protocol for Animal 2. Add 180 µl PBS and homogenize the sample
Tissues” from step 4. using an electric homogenizer or a disposable
microtube pestle.

26 Genomic DNA Purification

User-Developed Protocols
Isolation of genomic DNA from saliva
1. Ensure that the donor animal has not eaten in
the preceding 30 minutes. Collect 1 ml saliva.
2. Add 4 ml PBS (not provided) to the sample and
centrifuge at 1800 x g for 5 min.
3. Carefully decant the supernatant. Resuspend the
pellet in 180 µl PBS.
DNeasy spin columns copurify RNA and DNA
in parallel when both are present in the sample.
RNA may inhibit some downstream reactions,
but it does not inhibit PCR. If RNA-free genomic
DNA is required, 20 µl of RNase A stock
solution (20 mg/ml) should be added to the
sample before the addition of proteinase K.
4. Add 250 µl proteinase K solution and 200 µl
Buffer AL to the sample, mix thoroughly by
votexing, and incubate at 70°C for 10 min.
Continue with the “DNeasy Protocol for
Cultured Animal Cells” from step 3.
In order to ensure efficient lysis, it is essential
that the sample and buffer AL be mixed
immediately and thoroughly.
Isolation of genomic DNA from nails, hair, or bird
feathers*
1. Cut the sample into small pieces, place in a
1.5 ml microcentrifuge tube, and add 200 µl
Buffer X1. Incubate at 55°C for at least 1 h until
the sample is dissolved. Invert the tube
occasionally to disperse the sample, or place
on a rocking platform.
Buffer X1: 10 mM Tris·Cl; pH 8.0, 10 mM
EDTA, 100 mM NaCl, 40 mM DTT, 2% SDS,
250 µg/ml proteinase K. Add proteinase K and
DTT immediately before use.
* Feather quills will remain undissolved during step 1, therefore it will be
necessary to transfer the supernatant to a new microcentrifuge tube at
the end of step 1.
Genomic DNA Purification
2. Add 200 µl Buffer AL and 200 µl ethanol to the
sample and mix by vortexing.
3. Pipet the mixture from step 2 into a DNeasy
spin column placed in a 2 ml collection tube
(provided). Centrifuge at ≥6000 x g for 1 min.
Discard flow-through and collection tube.
4. Place the DNeasy column in a new 2 ml
collection tube (provided), add 500 µl
Buffer AW1, and centrifuge for 1 min at
≥6000 x g for 1 min. Discard flow-through
and collection tube.
5. Place the DNeasy column in a 2 ml collection
tube (provided), add 500 µl Buffer AW2, and
centrifuge for 3 min at full speed to dry the
DNeasy membrane. Discard flow-though and
collection tube.
6. Elute the DNA in 50–100 µl Buffer AE or
distilled water.
Elution in 50 µl will yield more concentrated
DNA, whereas elution in 100 µl will recover a
greater amount of DNA. If the expected amount
of DNA is not known, it is better to elute in
several aliquots of 50 µl, as these can be
combined if necessary. Elution of the DNA in
Buffer AE is recommended if the DNA is to be
stored, since DNA stored in water is subject to
acid hydrolysis.
27
 Isolation of DNA from compact bone 6. To 50 mg of pellet, add 360 µl Buffer ATL.

1. Completely remove bone marrow and soft
tissues using razor blades and/or sandpaper.
2. Crush the bone into small fragments. Grind to a
fine powder using a mixer mill or a metal
blender half-filled with liquid nitrogen.
3. Transfer 5 g powder into sterile 50 ml
polypropylene tubes and add 40 ml of 0.5 M
EDTA, pH 7.5, to decalcify the sample. Agitate
the tubes on a rotor at 4°C for 24 h.
4. Centrifuge the sample at 2000 x g for 15 min.
Discard the supernatant. Repeat the
decalcification process several times.
Generally, decalcification takes 3–5 days. The
decalcification process can be monitored by
adding a saturated solution of ammonium
oxalate, pH 3.0, to the decanted supernatant. If
the solution remains clear, the decalcification
process can be stopped.
7. Add 40 µl proteinase K, mix by vortexing, and
incubate at 55°C until the pellet is completely
lysed. Vortex occasionally during incubation to
disperse the sample, or place in a shaking
water bath or on a rocking platform.
8. Vortex for 15 s. Add 400 µl Buffer AL to the
sample, mix thoroughly by vortexing, and
incubate at 70°C for 10 min.
9. Add 400 µl ethanol (96–100%) to the sample
and mix thoroughly by vortexing.
10. Pipet up to 650 µl of the mixture from step 9
into the DNeasy column placed in a 2 ml
collection tube (provided). Centrifuge at
≥6000 x g. Discard flow-through and collection
tube. Repeat until all of the sample has been
loaded. Continue with the “DNeasy Protocol for
Animal Tissues” from step 6.

5. Wash the pellet with 40 ml sterile deionized

water to remove ions that have accumulated
during the decalcification. Centrifuge the
sample at 2000 x g for 15 min and discard
the supernatant. Repeat this washing procedure
3 times.

28 Genomic DNA Purification

References
1. Hirsch, H.H. and Bossart, W. (1999)
Two-centre study comparing DNA preparation
and PCR amplification protocols for herpes
simplex virus detection in cerebrospinal fluids
of patients with suspected herpes simplex
encephalitis. J. Med. Virol. 57, 31.
2. Miller, S.A., Dykes, D.D., and Polesky, H.F.
(1988) A simple salting-out procedure for
extracting DNA from human nucleated cells.
Nucleic Acids Res. 16, 1215.
3. Ausubel, F.M., Brent, R., Kingston, R.E., Moore,
D.D., Seidman, J.G., Smith, J.A., and Struhl, K.
(eds.) (1994) Current Protocols in Molecular
Biology, John Wiley & Sons, New York.
4. Sambrook, J. and Russell, D. (2001) Molecular
Cloning — A Laboratory Manual, 3rd Ed.,
Cold Spring Harbor Laboratory Press, New
York.
5. Heermann, K.H., Gerlich, W.H., Chudy, M.,
Schaefer S., and Thomssen, R. (1999)
Quantitative detection of hepatitis B virus DNA
in two international reference plasma
preparations. Eurohep Pathobiology Group.
J. Clin. Microbiol. 37, 68.
6. Verhagen, O.J., Wijkhuijs, A.J., van der
Sluijs-Gelling, A.J., Szczepanski, T., van der
Linden-Schrever, B.E., Pongers-Willemse, M.J.,
van Wering, E.R., van Dongen, J.J., and van
der Schoot, C.E. (1999) Suitable DNA isolation
method for the detection of minimal residual
disease by PCR techniques. Leukemia 13,
1298.
7. Reimann, U., Guntermann, D., and Weber, O.
(1998) High-throughput DNA purification with
DNeasy 96 — more than just mouse tails.
QIAGEN News 1998 No. 3, 7.
Genomic DNA Purification
8. Schwarz, H. (1997) Rapid high-throughput
purification of genomic DNA from mouse and
rat tails for use in transgenic testing. Technical
Tips Online
(http://www.elsevier.com/locate/tto) T01146.
9. Witzemann, V., Schwarz, H., Koenen, M.,
Berberich, C., Villaroel, A., Wernig, A.,
Brenner, H.R., and Sakmann, B. (1996)
Acetylcholine receptor e-subunit deletion causes
muscle weakness and atrophy in juvenile and
adult mice. Proc. Natl. Acad. Sci. USA 93,
13286.
10. Watson, A.J., Fuller, L.J., Jeenes, D.J., and
Archer, D.B. (1999) Homologs of aflatoxin
biosynthesis genes and sequence of aflR in
Aspergillus oryzae and Aspergillus sojaa.
Appl. Environ. Microbiol. 65, 307.
11. Luperchio, S.A., Newman, J.V., Dangler, C.A.,
Schrenzel, D., Brenner, D.J., Steigerwalt, A.G.,
and Schauer, D.B (2000) Citrobacter roden-
tium the causative agent of transmissible murine
colonic hyperplasia, exhibits clonality:
synonymy of C. rodentium and mouse-
pathogenic E. coli. J. Clin. Microbiol. 38, 4343.
12. Maeda, N., Palmarini, M., Murgia, C., and
Fan, H. (2001) Direct transformation of rodent
fibroblasts by jaagsiekte sheep retrovirus DNA.
Proc. Natl. Acad. Sci. USA 98, 4449.
13. Rowe-Magnus, D., Guerout, A-M., Ploncard, P.,
Dychinco, B., Davies, J., and Mazel, D. (2001)
The evolutionary history of chromosomal
super-integrons provides ancestry for
multiresistant integrons. Proc. Natl. Acad. Sci.
USA 98, 652.
29
 Ordering Information
Product Contents Cat. No.

DNeasy Tissue Kits — for DNA isolation from tissues, rodent tails, and cultured cells
DNeasy Tissue Kit (50) 50 DNeasy Spin Columns, Proteinase K, 69504
Buffers, Collection Tubes (2 ml)

DNeasy Tissue Kit (250) 250 DNeasy Spin Columns, Proteinase K, 69506
Buffers, Collection Tubes (2 ml)

DNeasy 96 Tissue Kits — for high-throughput DNA isolation from animal tissues and cells
DNeasy 96 Tissue Kit (4)* For 4 x 96 DNA minipreps: 4 DNeasy 96 Plates, 69581
Proteinase K, Buffers, S-Blocks, AirPore™ Tape Sheets,
Collection Microtubes (1.2 ml), Elution Microtubes RS,
Caps, 96-well Plate Registers

DNeasy 96 Tissue Kit (12)* For 12 x 96 DNA minipreps: 12 DNeasy 96 Plates, 69582
Proteinase K, Buffers, S-Blocks, AirPore Tape Sheets,
Collection Microtubes (1.2 ml), Elution Microtubes RS,
Caps, 96-well Plate Registers

Related products
QIAGEN Genomic-tip 20/G 25 columns 10223

QIAGEN Genomic-tip 100/G 25 columns 10243

QIAGEN Genomic-tip 500/G 10 columns 10262

Mixer Mill MM 300 — for efficient high-throughput disruption of biological samples

Mixer Mill MM 300, Universal laboratory mixer mill 85110
100-115V/50-60Hz (100/115 V, 50/60 Hz)

Mixer Mill Adapter Set (2 x 96)† 2 Sets of Adapter Plates and 2 racks for use 69999
with 1.5 or 2.0 ml microcentrifuge tubes on
the Mixer Mill MM 300

Tungsten Carbide Beads, Tungsten Carbide Beads, suitable for use 69997
3 mm (200)‡ with 1.2 ml Collection Microtubes

* Requires use of the QIAGEN 96-Well-Plate Centrifugation System.

†
Adapter sets are available exclusively from QIAGEN.
‡
Other disruption vessels and beads are available from Retsch (www.retsch.de).

30 Genomic DNA Purification

Ordering Information
Product Contents Cat. No.

96-well plate centrifugation system — for high-throughput purification procedures

Centrifuge 4-15C Universal laboratory centrifuge with brushless motor 81010
(120 V, 60 Hz) (120 V, 60 Hz)

Centrifuge 4K15C Universal refrigerated laboratory centrifuge with 81210

(220 V, 60 Hz) brushless motor (220 V, 60 Hz)

Plate Rotor 2 x 96* Rotor for 2 QIAGEN 96-well plates, 81031

for use with QIAGEN centrifuges

Accessories
Genomic DNA Buffer Set† Buffers including specific lysis buffers for yeast, 19060
bacteria, cells, blood, and tissue:
Y1, B1, B2, C1, G2, QBT, QC, QF;
for 75 mini-, 25 midi-, or 10 maxipreps

Buffer AW1 242 ml Wash Buffer (1) Concentrate for 1000 spin, 19081
(concentrate, 242 ml) 250 midi, or 100 maxi preps

Buffer AW2 (concentrate, 324 ml) 324 ml Wash Buffer (2) Concentrate 19072

Buffer AL (216 ml) 216 ml for 1000 preps 19075

Buffer ATL (200 ml) 200 ml Tissue Lysis Buffer for 1000 preps 19076

Buffer AE (240 ml) 240 ml Elution Buffer for 1000 preps 19077

QIAGEN Proteinase K (2 ml) 2 ml (>600 mAU/ml, solution) 19131

QIAGEN Proteinase K (10 ml) 10 ml (>600 mAU/ml, solution) 19133

Collection Tubes (2 ml) 1000 Collection Tubes (2 ml) 19201

Collection Microtubes and Caps Nonsterile polypropylene tubes (1.2 ml), 120007
2304 in packs of 96, and nonsterile polypropylene
caps for Collection Microtubes

Elution Microtubes RS Nonsterile polypropylene tubes (0.6 ml); 120008

2304 in racks of 96. Includes caps

* The Plate Rotor 2 x 96 is available exclusively from QIAGEN.

†
Enzymes must be purchased separately.

Genomic DNA Purification 31

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