genomic dna purification technical hints, applications, and protocols

Genomic DNAPurification

Technical hints, applications,

and protocols

Trademarks and disclaimers

Patented or patent-pending and/or registered or registration-pendingtrademarks of the QIAGEN Group: QIAGEN®, AirPore™, DNeasy®,Masscode™, QuantiTect™.

ABI PRISM is a registered trademark of Applera Corporation or itssubsidiaries.

Black Hole Quencher and BHQ are trademarks of BiosearchTechnologies, Inc. (BTI).

Triton is a registered trademark of Rohm & Haas Inc.

The PCR process is covered by U.S. Patents 4,683,195 and 4,683,202and foreign equivalents owned by Hoffmann-La Roche AG.

Purchase of QIAGEN products for PCR is accompanied by a limitedlicense to use them in the Polymerase Chain Reaction (PCR) process forresearch and development activities in conjunction with a thermal cyclerwhose use in the automated performance of the PCR process is coveredby the up-front license fee, either by payment to Applied Biosystems oras purchased, i.e. an authorized thermal cycler. The PCR process iscovered by U.S. Patents 4,683,195 and 4,683,202 and foreignequivalents owned by Hoffmann-La Roche AG.

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

© 2002 QIAGEN, all rights reserved.

2 Genomic DNA Purification

Genomic DNA Purification 3

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 6Animal, yeast, and bacterial cell cultures 6Animal blood 7

Amount of starting material 7DNA yields 7

Disruption and lysis 8

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

DNA isolation methods 10

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

Applications using DNeasy Tissue Kits 14

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

Protocols 22

User-Developed Protocols 27

References 29

Ordering Information 30

IntroductionOver the last decade there has been an increased requirement forisolation of pure genomic DNA that performs well in any down-stream application. Such downstream applications include PCR(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 sourcesare becoming increasingly diverse. Each has its own difficultiesassociated with the isolation of pure genomic DNA.

For many analysis techniques, DNA quality is the single mostimportant factor. Poor quality DNA can lead to suboptimal results,and DNA that is impure or contaminated will not perform well indownstream applications. The choice of sample preparation methoddirectly affects the results of the downstream application.

QIAGEN offers extensive expertise in the purification of nucleicacids. This guide gives an overview of the techniques used for isolationof DNA from a wide variety of animal tissue and cell sources, aswell as guidelines for successful downstream applications. Furthergeneral information on the handling and analysis of genomic DNA isavailable in the QIAGEN® Bench Guide. QIAGEN also offers guidesfor plant tissues and clinical samples. Please see the brochure PlantNucleic Acid Purification, for plants and fungi, and the Applicationand Product Guide for Molecular Diagnostics, for human clinicalsamples. All QIAGEN literature is available free on request fromQIAGEN Technical Services, or can be downloaded from our website: www.qiagen.com/literature/brochures/index.asp.

Effects of contaminants on downstream applications

Carryover of contaminants such as salts, phenol, ethanol, anddetergents from conventional purification procedures can inhibitperformance of DNA in downstream applications. DNA preparedusing DNeasy® technology is free from contaminants, ensuringconsistently good performance in downstream applications (Figures 1 and 2).

Researchers require reliable purification methods that minimize therisk of DNA contamination, facilitating high-quality results indownstream applications. There is also a need for purificationmethods to be fast, safe, robust, and easy to handle. Manyresearchers also want the added benefit of being able to use onecommercial kit for many sample types.

Home-made methods do not fulfill all the above requirements. Inaddition, they often adversely affect reproducibility, whereascommercial kits offer a proven and guaranteed solution. DNeasyTissue Kits fulfill all the above purification requirements andguarantee high-quality DNA with every prep.


DNA Contamination Leads to Lower Yieldsand Quality of PCR Products

Figure 1. PCR amplification of genomic DNA purifiedusing the DNeasy Tissue Kit in the absence (DNeasy) andpresence of increasing concentrations of indicatedimpurities shows that impure DNA preparations lead tofailure of PCR and/or low yields of amplicons. From topleft: 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.

M 0.2 0.5%Phenol

NaCl Ethanol

SDSM

M M

DNea

sy

DNea

sy

DNea

sy

DNea

sy

0.005 0.01%

25 50 mM 1 5%

Starting materialsGenomic DNA can be isolated from a wide variety of startingmaterials, for many different fields of study. These include:

◆ Cultured animal cells◆ Animal tissues (e.g., brain, liver, spleen, lung, heart, and kidney)◆ Crude lysates of any animal tissue◆ Animal blood◆ Buffy coats (from animal blood)◆ Animal bone marrow◆ Fixed tissues◆ Yeast◆ Bacteria◆ Insects◆ Rodent tails

DNA is a relatively stable molecule. However, introduction ofenzymatically 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 shouldtherefore be stored at a slightly alkaline pH, e.g., in TE buffer or inBuffer AE from QIAGEN.

Degradation of DNA has a major effect on any results obtained,generating errors that are both quantitative and qualitative. Forexample, a reduction in DNA size may lead to the failure ofdownstream applications such as PCR-based applications andhybridization. This is especially important in areas where DNAdegradation 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 collectionand storage of tissue samples before DNA isolation. For plant tissuesand clinical samples, please see the brochures Plant Nucleic AcidPurification, and the Application and Product Guide for MolecularDiagnostics. Both brochures are available free on request.


Effect of Contamination on Spectrophotometry

Figure 2. Effect of phenol on DNA quantification. UV scanof Q (QIAGEN): NA purified using silica-gel–membranetechnology (100 µl sample); P (phenol): The same samplewith 1 µl phenol diluted 1:1000 in water (final dilution1:100,000). Phenol contamination of DNA can interferewith spectrophotometric readings and lead to over-estimation.

0.4

0.0220 320

Abs

orba

nce

Wavelength (nm)

DNA +phenol (P)

DNA (Q)

Sample collection and storage before isolation of genomic DNA

The quality of the starting material affects the quality and yield of theisolated DNA. Optimal results are obtained with fresh material, orwith material that has been immediately frozen (frozen in liquidnitrogen or in a mixture of ethanol and dry ice) and stored at –20°Cor –70°C. Repeated freezing and thawing of stored samples shouldbe avoided, as this leads to reduced fragment size and precipitationof the DNA, and in clinical samples, to reduced yields of pathogenDNA (e.g., viral DNA). Use of poor quality starting material will alsolead to reduced length and yield of purified DNA. In general,genomic DNA yields will decrease if samples are stored at either2–8°C or –20°C without previous treatment.

The recommendations for storage of different starting materials arediscussed below. In some cases, storage under optimal conditionsmay not be possible, e.g., for preserved tissues. In such cases thedownstream application may be adjusted to compensate for poor orinappropriate 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 bestored in Buffer ATL, after proteinase K digestion, for up to 6 monthsat ambient temperatures without any reduction in DNA quality.Animal and human tissues can also be fixed for storage. Werecommend using fixatives such as alcohol and formalin; however,long-term storage of tissues in formalin will result in chemicalmodification of the DNA, which decreases DNA quality and resultsin shorter fragments upon purification. Fixatives that cause cross-linking, such as osmic acid, are not recommended if DNA will beisolated from the tissue. It is also possible to isolate DNA fromparaffin-embedded tissue.

Animal, yeast, and bacterial cell cultures

Cell cultures are usually harvested by centrifugation followed byremoval of the supernatant. Cells are then stored at –20°C or –70°C.Alternatively, animal cell nuclei can be prepared and stored at–20°C. For certain bacterial and yeast cultures that accumulate largeamounts of metabolites and/or form very dense cell walls, cellsshould be harvested in the early log phase of growth, and washedwith fresh medium. Fresh or frozen cell pellets can be used.


Table 1. Maximum recommended amounts ofstarting material*

Animal tissue 25 mg

Mouse tail 0.6–1.2 cm

Rat tail 0.6 cm

Cultured cells 5 x 106 cells

Bacteria 2 x 109 cells

Yeast 5 x 107 cells

Table 2. Sizes and molecular weights ofvarious genomic DNAs

Base pairs per Organism haploid genome

Escherichia coli K12 4.6 x 106

Saccharomyces cerevisiae 1.2 x 107

Dictyostelium discoideum 5.4 x 107

Arabidopsis thaliana 1.2 x 108

Caenorhabditis elegans 9.7 x 107

Drosophila melanogaster 1.8 x 108

Gallus domesticus (chicken) 1.2 x 109

Mus musculus (mouse) 3.0 x 109

Rattus norvegicus (rat) 3.0 x 109

Xenopus Iaevis 3.1 x 109

Homo sapiens 3.3 x 109

* Using DNeasy Tissue procedures.

Animal blood

Freshly drawn animal whole blood (nucleated or non-nucleated)should be digested with proteinase K. The amount of blood used iscritical and tenfold less nucleated blood should be used in theprocedure compared with non-nucleated blood. Neither type ofblood should be coagulated. Buffer AL should be added to bloodcontaining proteinase K and incubated for 10 minutes at 70°C fordigestion. The appropriate DNeasy Tissue Kit protocol should then befollowed.

Amount of starting material

The amount of starting material to use is a key point in the successfulisolation of genomic DNA. Starting sample size depends on thetissue or cells being used, but the amounts shown in Table 1 (page 6)are a good guide when using DNeasy Tissue procedures. Often, anexcess of starting material can be counter-productive, and as a gen-eral guide “less is more”. If you have no information on DNA contentand your starting material is not shown in Table 1, we recommendbeginning with half the maximum amount of starting material indicat-ed in Table 1. Depending on the yield obtained, the sample size canbe increased in subsequent preparations.

DNA yields

The DNA content of cells is not homogeneous, and depends on thetype of cell and the size of the genome. Genome sizes can vary bya factor of 1000, e.g., between bacterial (106 bp) and animal (109 bp) cells (Table 2, page 6). Animal cells usually have genomessized 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 thereforemore DNA per milligram of tissue can be expected. For samples withvery high DNA content e.g., spleen or cell lines with a high degreeof ploidy, use less than the recommended amount of starting materiallisted in Table 1. Depending on the yield obtained, the sample sizecan be increased in subsequent preparations.

Purification of DNA from very small amounts of starting material isalso possible. If the sample has less than 5 ng DNA (<10,000genome copies), carrier DNA (a homopolymer such as poly dA, poly dT, or gDNA) should be added to the starting material. It isimportant to check that the carrier DNA does not interfere with yourdownstream application.

Yields of DNA obtained using DNeasy Tissue technology are shownin Table 3.


Table 3. Yields of genomic DNA with DNeasyTissue Kits

Yield

Total nucleic Source acids (µg)* DNA (µg)†

Lymphocytes 20–30 15–25(5 x 106)

HeLa cells 40–60 15–25(2 x 106)

Liver (25 mg) 60–115 10–30

Brain (25 mg) 35–60 15–30

Lung (25 mg) 8–20 5–10

Heart (25 mg) 25–45 5–10

Kidney (25 mg) 40–85 15–30

Spleen (10 mg) 25–45 5–30

Mouse tail, 1.2 cm 15–30 10–25(tip section)

Rat tail, 0.6 cm 25–60 20–40(tip section)

* Nucleic acids purified without RNase treatment.† Nucleic acids purified with RNase treatment.

Disruption and lysisComplete disruption and lysis of cell walls and plasma membranes of cells and organelles is essential for all genomic DNA isolationprocedures. Incomplete disruption results in significantly reducedyields. The methods listed below are suitable for the breakdown ofall cellular and organellar membranes. The DNA obtained will varyin size between these methods due to differences in shearing appliedto the genomic DNA during lysis. It should be noted that someprotocols require prior isolation of cell nuclei, and will therefore yieldtotal 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 adetergent (for breaking down cellular membranes) and a protease(for digestion of protein cellular components). The choice of proteasedepends on the lysis buffer used. Both QIAGEN Proteinase K andQIAGEN Protease have high activity in buffers commonly used forDNA isolation. QIAGEN Proteinase K is recommended for bufferscontaining SDS and >8 mM EDTA.

Enzymatic lysis of bacteria and yeast

Additional enzymatic lysis should be used when isolating genomicDNA from Gram-positive bacteria or yeast. The structure of theGram-negative bacterial cell wall means that additional enzymaticlysis is unnecessary for these species. Lysozyme (for Gram-positivebacteria) or lyticase (for yeast) should be added to the enzymaticlysis buffer immediately before use.

Disruption using rotor-stator homogenizers

Rotor-stator homogenizers thoroughly disrupt animal tissues inapproximately 5–90 seconds depending on the toughness of thesample. The rotor turns at very high speed disrupting the sample bya combination of turbulence and mechanical shearing. Rotor-statorhomogenizers are available in different sizes and have probes ofdifferent sizes. Probes with diameters of 5 mm and 7 mm aresuitable for volumes up to 300 µl and can be used in microcentrifugetubes. Probes with a diameter of 10 mm or greater require largertubes. This method can cause minor shearing of the genomic DNAand may have a bearing on its molecular weight.


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

Disruption using the Mixer Mill MM 300 and other bead mills

Disruption using a bead mill involves agitation at high speed in thepresence of beads. Disruption occurs by the shearing and crushingaction of the beads as they collide with the cells. Disruption efficiencyis influenced by the factors shown in Table 4 (page 8). The optimaltypes of beads to use for disruption of various starting materials inthe Mixer Mill MM 300 are shown in Table 5.

Glass beads should be pretreated before use by washing inconcentrated nitric acid. Alternatively, commercially available acid-washed glass beads can be used. All other disruptionparameters should be determined empirically for each application.

Samples can be disrupted in either an appropriate buffer or in liquidnitrogen. If disruption is performed in liquid nitrogen, it is critical toensure that the sample remains frozen at all times. Disruption usingliquid nitrogen in conjunction with a bead mill is comparable to thatachieved using a pestle and mortar (see below). If disruption isperformed in buffer, it is important to optimize the disruption time. If disruption is prolonged once material has been lysed, significantdegradation can occur.

Disruption using a mortar and pestle

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


Table 5. Starting material and optimal beads to use

Mean bead sizeStarting sample Bead type (diameter)

Animal tissues Stainless steel 3–7 mm

Animal cells Glass 0.5 mm

Bacteria Glass 0.1 mm

Yeast Glass 0.5 mm

Figure 3. The Mixer Mill MM 300 allows processing of up to 192 tissue samples in just 2–4 minutes.

Efficient High-Throughput Disruption of Tissue Samples

DNA isolation methodsMany different methods and technologies are available for theisolation of genomic DNA. In general, all methods involve disruptionand lysis of the starting material followed by the removal of proteinsand other contaminants and finally recovery of the DNA. Removal ofproteins is typically achieved by digestion with proteinase K,followed by salting-out, organic extraction, or binding of the DNA toa solid-phase support (either anion-exchange or silica technology).DNA is usually recovered by precipitation using ethanol orisopropanol. The choice of a method depends on many factors: therequired quantity and molecular weight of the DNA, the purityrequired 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 methodsexist (a comparison of methods is shown in Figure 7, page 13).Home-made methods often work well for researchers who havedeveloped and regularly use them. However, they usually lackstandardization and therefore yields and quality are not alwaysreproducible. Reproducibility is also affected when the method isused by different researchers, or with different sample types.

The separation of DNA from cellular components can be divided intofour stages:

1. Disruption2. Lysis3. Removal of proteins and contaminants4. 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 celllysates at high temperatures (e.g., 90°C for 20 minutes), or toperform a proteinase K digestion, and then use the lysates directly indownstream applications. Considered ”quick-and-dirty” techniques,these methods are only appropriate for a limited range ofapplications. The treated lysate usually contains enzyme-inhibitingcontaminants, such as salts, and DNA is often not at optimal pH (1).Furthermore, incomplete inactivation of proteinase K can result infalse negative results and high failure rates. It is not recommended tostore DNA prepared using this method, as the high levels ofcontamination often result in DNA degradation.


Salting-out methods

Starting with a crude lysate, ”salting-out” is another conventionaltechnique where proteins and other contaminants are precipitatedfrom the cell lysate using high concentrations of salt such aspotassium acetate or ammonium acetate (2). The precipitates areremoved by centrifugation, and the DNA is recovered by alcoholprecipitation. Removal of proteins and other contaminants using thismethod may be inefficient, and RNase treatment, dialysis, and/orrepeated alcohol precipitation are often necessary before the DNAcan be used in downstream applications. DNA yield and purity arehighly variable using this method.

Organic extraction methods

Organic extraction is a conventional technique that uses organicsolvents to extract contaminants from cell lysates (3, 4). The cells arelysed using a detergent, and then mixed with phenol, chloroform,and isoamyl alcohol. The correct salt concentration and pH must beused during extraction to ensure that contaminants are separated intothe organic phase and that DNA remains in the aqueous phase.DNA is usually recovered from the aqueous phase by alcoholprecipitation. This is a time-consuming and cumbersome technique.Furthermore, the procedure uses toxic compounds and may not givereproducible yields (5). DNA isolated using this method may containresidual phenol and/or chloroform, which can inhibit enzyme reactionsin downstream applications, and therefore may not be sufficientlypure for sensitive downstream applications such as PCR (6). Theprocess also generates toxic waste that must be disposed of withcare and in accordance with hazardous waste guidelines. In addition,this technique is almost impossible to automate, making it unsuitablefor high-throughput applications.

Cesium chloride density gradients

Genomic DNA can be purified by centrifugation through a cesiumchloride (CsCl) density gradient. Cells are lysed using a detergent,and the lysate is alcohol precipitated. Resuspended DNA is mixedwith CsCl and ethidium bromide and centrifuged for several hours.The DNA band is collected from the centrifuge tube, extracted withisopropanol to remove the ethidium bromide, and then precipitatedwith ethanol to recover the DNA. This method allows the isolation ofhigh-quality DNA, but is time consuming, labor intensive, andexpensive (an ultracentrifuge is required), making it inappropriate forroutine use. This method uses toxic chemicals and is also impossibleto automate.


Anion-exchange methods

Solid-phase anion-exchange chromatography is based on theinteraction between the negatively charged phosphates of the nucleicacid and positively charged surface molecules on the substrate. DNAbinds to the substrate under low-salt conditions, impurities such asRNA, cellular proteins, and metabolites are washed away usingmedium-salt buffers, and high-quality DNA is eluted using a high-saltbuffer. The eluted DNA is recovered by alcohol precipitation, and issuitable for all downstream applications.

Anion-exchange technology completely avoids the use of toxicsubstances, and can be used for different throughput requirements aswell as for different scales of purification. The isolated DNA is sizedup to 150 kb, with an average length of 50–100 kb. QIAGENoffers QIAGEN Genomic-tips for the purification of high-molecular-weight DNA.

Silica-based methods — DNeasy Tissue Kits

DNeasy Tissue technology provides a simple, reliable, fast, andinexpensive method for isolation of high-quality DNA. This method isbased on the selective adsorption of nucleic acids to a silica-gelmembrane in the presence of high concentrations of chaotropic salts(Figure 4). Use of optimized buffers in the lysis procedure ensuresthat only DNA is adsorbed while cellular proteins, and metabolitesremain in solution and are subsequently washed away. This is sim-pler and more effective than other methods where precipitation orextraction is required. Ready-to-use DNA is then eluted from thesilica-gel membrane using a low-salt buffer. No alcohol precipitationis required, and resuspension of the DNA, which is often difficult ifthe DNA has been over-dried, is not required.

DNeasy Tissue Kits are designed for rapid isolation of pure totalDNA (genomic, viral, and mitochondrial) from a wide variety ofsample sources, including fresh and frozen animal cells and tissues,yeasts, and blood. DNA purified using DNeasy Tissue Kits is freefrom contamination and enzyme inhibitors and is highly suited forapplications such as Southern blotting, PCR, real-time PCR, RAPD,RFLP, and AFLP analyses. DNeasy Tissue Kits are available inconvenient spin-column or 96-well formats, suitable for a wide rangeof 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 thislength is particularly suitable for PCR analysis as well as Southernblotting analysis (7–13). Silica-gel spin technology is not suitable ifgenomic DNA >50 kb is required for certain cloning or blottingapplications. QIAGEN recommends the use of QIAGEN Genomic-tipsfor these applications.


Lyse

Tissue sample

Bind DNA

Wash

Ready-to-use DNA

Elute

Mouse tail or animal tissue

Collectmouse tails ortissue samplesand lyse

Bind DNA

Wash

Elute into ElutionMicrotubes RS

Ready-to-use DNA

DNeasy Tissue Spin and 96-Well Plate Procedures

Figure 4. The DNeasy Tissue spin and 96-well plate procedures.

The DNeasy Tissue procedure is suitable for both very small andlarge sample sizes, from as little as 100 cells up to 5 x 106 cells. Inorder to obtain optimal DNA yield and quality, it is important not tooverload the DNeasy System, as this can lead to significantly loweryields than expected (Figure 5). Overloading the DNeasy System canalso adversely affect the purity of the DNA (Figure 6).

The DNeasy Tissue procedure is also highly suited for purification ofDNA from very small amounts of starting material. If the sample hasless than 5 ng DNA (<10,000 copies), 3–5 ng carrier DNA (a homopolymer such as poly dA, poly dT, or gDNA) should beadded to the starting material. Ensure that the carrier DNA does notinterfere with the downstream application.


Figure 5. DNA was purified from tissue samplespooled from 10 mice using the DNeasy Tissue protocolfor rodent tails. DNA yield was determined spectro-photometrically. Each point represents the mean andstandard deviation from 10 preparations.

Figure 6. The DNA purity of the samples described inFigure 5 was determined spectrophotometrically bymeasuring the A260/A280 ratio.

Figure 7. Length of time taken for the common methods of genomic DNA isolation, after proteinase K digestion. Simple methods are defined as boiling preps and otherprotocols that do not include a lysis step. Alcohol precipitation is the precipitation of proteinase K-digested lysates after removal of insoluble particles by centrifugation. Fororganic extraction phenol/chloroform is added to the proteinase K-digested lysate,vortexed and centrifuged. The upper phase is then subject to alcohol precipitation.Salting-out is defined as treatment of the proteinase K-digested lysate with a high-saltbuffer. This is incubated and the proteins precipitated by centrifugation. The supernatantis then subject to alcohol precipitation. See pages 10–12 for further information on DNAisolation methods.

* No proteinase K digestion.

10

0

20

30

40

50

10 20 3015 25

Tissue (mg)

LiverSalivary glandKidney

LiverSalivary glandKidney

1.8

1.7

1.9

2.0

2.1

10 20 3015 25

A26

0/A

280

ratio

Tissue (mg)

Effect of Sample Size on DNA Yield

DNA Purity and Time Required for Different Isolation Methods

Effect of Sample Size on DNA Purity

QIAGENDNeasy

Tissue Kits

Time

alcoholprecipi-tation

High

Low

QIAGENAnion-

exchangetechnology

Alcoholprecipitation

Simplemethods*

1 x CsClgradient

Purit

y

Less More

Organicextraction

Salting-out

DN

A y

ield

(µg)

Applications using DNeasy Tissue KitsDNeasy Tissue and DNeasy 96 Tissue Kits provide simple, rapid,and reproducible methods for the isolation of DNA from a widerange of sample sources. DNA purified using DNeasy 96 Tissue issuitable 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 thefollowing pages. This section contains many applications currentlyperformed using DNeasy Tissue Kits. It provides a guide to the rangeof uses of DNeasy Tissue technology.

Functional genomics

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


Figure 8. Mice with a nonfunctional CRALBP (cellular retinaldehyde binding protein)gene (Rlbp1-/Rlbp1- mice) were generated. In humans, mutations in the CRALBP genecause retinal pathology and delayed dark adaptation. The primary biochemical role ofthis gene is unknown, and knockout mice were generated to address this question. TheDNeasy Tissue Kit was used to purify DNA from tail tips taken from the offspring of micechimeric for a nonfunctional version of the CRALBP gene. Primers were designed toamplify either wild-type (wt) or targeted (neo1 and neo2) alleles, and used to amplifyDNA from mice of interest. This method yielded high-quality DNA that gave reliableresults 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., andCrabbs, J. W. (2001) Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice resultsin delayed dark adaptation. Neuron 29, 739. Published with permission.

wt neo1

neo2

wt neo1

neo2

Identification of Nonfunctional Genes by PCR

1018

506

+/+ –/–

396


Figure 9. The role of the Bax protein in nerve cell death, and the response of centralnervous system cells to oxidative stress was examined in knockout mice. Bax-homozygous knockout mice were bred by crossing Bax-heterozygous mice, so that acolony and a source of embryos lacking the Bax protein were maintained. GenomicDNA was isolated from pups using the DNeasy Tissue Kit and the genotype of individualmice 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 Knockout Animals Using DNeasy Tissue Technology

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

Genotyping of Transgenic Tadpoles by PCR

– 500 bp

– 500 bp– 300 bp

Nocco

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GFPco

ntrol

M

Noc → – 250 bp

1 2 3 4 5 6 7

1 2 3

– + + + + + +

GFP →

Molecular breeding

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


Microsatellite Analysis of Farmed Atlantic Salmon

Figure 11. Genetic profiling of farmed Atlantic salmon (Salmo salar) was performed inconjunction with a breeding program aimed at improving performance of stocks,maintaining and maximizing genetic variation, and determining parentage, thuspreventing inbreeding. Total DNA was isolated from 5–10 mg Atlantic salmon fin tissuestored in ethanol, using the standard "DNeasy 96 Tissue Protocol for High-ThroughputDNA 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 2of the protocol. In step 15, plates were centifuged for 15 minutes to ensure completeethanol 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.

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Genetic studies in bacteria and yeast

The study of bacteria is a diverse field, with far-reaching implicationsfor human health and disease, animal husbandry, agriculture andaquaculture, the environment, and the food and brewing industries.Studies at the genetic level in bacteria and yeast have led tosignificant advances in these areas.


RFLP Analysis of Borrelia burgdorferi DNA

Figure 12. Total bacterial DNA was isolated from B. burgdorferi, the causative agent ofLyme disease, using the DNeasy Tissue Kit. Possible variations in bdr-flanking regionswere assessed using an RFLP-based approach. RFLP analysis indicating stability of bdr-flanking regions. Southern blots of total B. burgdorferi DNA digested with XbaI wereprobed 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 thatrecombinatorial variation of bdr genes is not essential for persistent mammalianinfection. These loci could provide new tools for plasmid profiling and strain typing, andcould be especially useful for quickly assessing possible variations of complex Borreliapopulations 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 invivo. Infection and Immunity 68, 1727. Published with permission.

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Disease research

Studies in human or animal cell lines and the use of animal modelshas led to a greater understanding of human diseases and theirtreatment. DNeasy Tissue technology is highly suited for thepurification of high-quality genomic DNA from a wide range of celllines and animal tissues.


Undifferentiated FLEC in Post-Transplantation Livers

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

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Figure 13. Adult rats were treated with retrorsine to prevent hepatocyte proliferation, andthen, in conjunction with a partial hepatectomy (PH) fetal liver epithelial cells (FLEC) weretransplanted 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 therat sry gene, located on the Y chromosome in transplanted cells, determined whetherundifferentiated FLEC remained after transplantation. It was demonstrated that largenumbers of undifferentiated FLEC (and their progeny) were present in livers that hadundergone a partial hepatectomy, while only small numbers remained in livers that werefunctioning 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.

Figure 14. DNA was purified using the DNeasy Tissue Kit. A chromosomal translocation in the Burkitt’s lymphoma cell lineRamos was detected using real-time PCR. Various numbers (7, 70, 700, and 7000 copies) of the tumor-specific translocationwere spiked into 250 ng translocation-free human genomic DNA (also purified using the DNeasy Tissue Kit). All quantitieswere 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 theABI PRISM® 7700 Sequence Detection System. Dual-labeled probes for the 5'–3' nuclease assay contained either A: 6-FAMas the reporter and Black Hole Quencher™1 (translocation-specific probe) or B: TAMRA as the reporter and Black HoleQuencher 2 (GAPDH-specific probe). The coefficient of determination (R2) for A was 0.9997.

A B

Copies of tumor-specific translocation7707007000

Natural history applications

Old dried skins, skeletons, and formalin-fixed, ethanol-preservedanimal 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 specimensare many. Bones have been found to be a much more reliable sourceof DNA, especially of long DNA fragments.


Successful Amplification of DNA from 8- and 19-Year-Old Bat Specimens

Figure 15. Small bones (a few millimeters long) and skin from fruit bats of the genusSturnira were used. The DNeasy Tissue protocol was modified to include additionalwashing and rehydration of tissues, prolonged proteinase K digestion, adjustment of pHbefore adsorption of DNA onto the DNeasy spin column, and reduction in the elutionvolume. This provided amplifiable cytochrome b DNA suitable for PCR. The data showsamplification of cytochrome b from an 8-year-old bat museum specimen preserved informalin/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 reverseprimers, each yielding a product of successively greater length. Reverse primers andapproximate 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 bonesdigested 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 templatewas 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 mammalmuseum specimens as a reliable source of DNA. BioTechniques 30 734. Published with permission.

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Phylogeny and biodiversity studies

Phylogenetic studies are essential for understanding evolutionaryhistory and species diversity. Furthermore, where species areendangered, phylogenetic analysis provides a valuable insight to theconservation risks to these species. Mouse lemurs are the world’ssmallest living primates and are native to Madagascar. Detailedinvestigation of species diversity for mouse lemurs (genusMicrocebus) is essential for evaluating the above factors to theseorganisms.


Phylogenetic Diversity in Madagascan Mouse Lemurs

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Figure 17. Genomic DNA from liver, spleen, kidney, or ear punches from individualsfrom an extensive array of localities was isolated using DNeasy Tissue Kits and was usedto examine the species diversity of Microcebus at the genetic level. Several mitochondrialDNA markers (mtDNA) were selected in order to show genetic variation in the inter- andintraspecific level. The figure shows the phylogeny derived from sequence alignment of2404 bp of combined mtDNA sequences from the control region homologous with thehypervariable region 1 in humans, COII and cytochrome b. Clades are color-coded toemphasize species diversity. The analysis showed unexpectedly high levels of speciesdiversity 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., andGanzhorn, J.U. (2000) Remarkable species diversity in Malagasy mouse lemurs (primates Microcebus) Proc. Natl.Acad. Sci. USA. 97, 11325. Published with permission.

Detection of viral pathogens

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


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

Figure 18. The presence of porcine circovirus type 2 (PCV2) in the semen of infectedboars was investigated. Four boars were infected with PCV2 and 2 boars wereinoculated with non-infected cells as controls. The DNeasy Tissue Kit was used to isolateDNA from serum and semen samples for several days post-infection (dpi), and PCV2nucleic acid was detected using PCR and nested PCR. Following infection, PCV2 DNAwas detected in semen concurrently with the presence of PCV2 DNA and antibodies inserum, and PCV2 was shed intermittently in the semen of infected boars. Controls fromleft 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 ofshedding in porcine circovirus type 2 in boar semen. J. Clin. Microbiol. 38, 4629. Published with permission.

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ProtocolsThe standard DNeasy Tissue protocols for purification of DNA fromrodent tails, animal tissues, and cultured cells, are detailed here (seealso the DNeasy Procedure flowchart on page 12). DNeasy Tissuetechnology is very versatile and can also be successfully used toisolate 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 spincolumn technology, DNeasy Tissue technology is also available in aconvenient high-throughput (96-well plate) format.

In addition, protocols are listed for the isolation of genomic DNAfrom saliva, nails, hair, or bird feathers, and from compact bone.These are User-Developed Protocols, developed by customers for theirown applications. QIAGEN is continually developing and optimizingDNeasy Tissue protocols for new sample sources, which are notlisted here.

Current standard and User-Developed protocols are always availablefrom QIAGEN Technical Service. Please contact QIAGEN TechnicalServices or your local distributor to receive free copies of any ofthese protocols or to inquire about other applications.

DNeasy Tissue and DNeasy 96 Tissue Handbooks contain all thestandard DNeasy protocols, and all standard and a selection ofUser-Developed Protocols are available to download, view, and print at www.qiagen.com/literature/protocols.

QIAGEN customers are a major source of information regardingadvanced or specialized uses of our products. This information ishelpful to other scientists as well as to the researchers at QIAGEN.Please contact us if you have any suggestions about productperformance or new applications and techniques.



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 piecesfor efficient lysis.

2. Add 20 µl proteinase K, mix by vortexing, andincubate at 55°C until the tissue is completelylysed.

Vortex occasionally during incubation todisperse the sample, or place in a shakingwater bath on a rocking platform.

Lysis time varies depending on the type of tissuebeing 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 andkidney contain high levels of RNA, which willcopurify with genomic DNA. If RNA-freegenomic DNA is required, carry out this step.

3. Vortex for 15 s. Add 200 µl Buffer AL to thesample, mix thoroughly by vortexing, andincubate 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 ofethanol. It is essential to apply all of theprecipitate to the DNeasy spin column.

5. Pipet the mixture from step 4 into the DNeasyspin column placed in a 2 ml collection tube(provided). Centrifuge at ≥6000 x g (8000 rpm)for 1 min. Discard flow-through and collectiontube.

6. Place the DNeasy spin column in a new 2 mlcollection 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 mlcollection tube (provided), add 500 µl Buffer AW2, and centrifuge for 3 min at full

speed to dry the DNeasy membrane. Discardflow-through and collection tube.

This step ensures that no residual ethanol iscarried over during the following elution.Remove the spin column carefully to ensure thatthe 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 theDNeasy 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 µlBuffer AE as this will cause the eluate to comeinto contact with the DNeasy spin column.

DNeasy protocol for rodent tails

1. Cut one (rat) or up to two (mouse) 0.4–0.6 cmlengths of tail into a 1.5 ml microcentrifugetube. Add 180 µl Buffer ATL. Earmark theanimal appropriately.

A maximum of 1.2 cm (mouse) or 0.6 cm (rat)tail should be used. When purifying the DNAfrom the tail of an adult mouse or rat, it isrecommended to use only 0.4–0.6 cm.

2. Add 20 µl proteinase K, mix by vortexing, andincubate at 55°C until the tissue is completelylysed. Vortex occasionally during incubation todisperse the sample, or place the tube in ashaking water bath on a rocking platform.After mixing the tail section with proteinase K,ensure the tail section is fully submerged. Lysisis usually complete in 6–8 h, but samples canbe 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, whichwill be copurified. RNase digestion can beused to destroy any residual RNA.


3. Vortex for 15 s. Add 400 µl Buffer AL–ethanolmixture to the sample, and mix thoroughly byvortexing.

A white precipitate may form on addition ofethanol. It is essential to apply all of theprecipitate to the DNeasy spin column.

4. Pipet the mixture from step 3 into the DNeasyspin column placed in a new 2 ml collectiontube (provided). Centrifuge at ≥6000 x g(8000 rpm) for 1 min. Discard flow-throughand collection tube.

5. Place the DNeasy spin column in a new 2 mlcollection 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 mlcollection tube (provided), add 500 µl Buffer AW2, and centrifuge for 3 min at fullspeed to dry the DNeasy membrane. Discardflow-through and collection tube.


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 theDNeasy membrane. Incubate at roomtemperature 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 orseparate tubes. Do not use more than 200 µlBuffer AE as this will cause the eluate to comeinto 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).

When using a frozen cell pellet, before addingPBS allow cells to thaw until the pellet can bedislodged by gently flicking the tube.

Optional: If RNA-free genomic DNA isrequired, add 4 µl RNase A (100 mg/ml) andincubate for 2 min at room temperature.

2. Add 20 µl proteinase K and 200 µl Buffer AL tothe sample, mix thoroughly by vortexing, andincubate at 70°C for 10 min. Do not addproteinase K directly to Buffer AL.

3. Add 200 µl ethanol (96–100%) to the sampleand mix thoroughly by vortexing. A whiteprecipitate may form on addition of ethanol. Itis essential to apply all of the precipitate to theDNeasy spin column.

4. Pipet the mixture from step 3 into the DNeasyspin column placed in a new 2 ml collectiontube (provided). Centrifuge at ≥6000 x g(8000 rpm) for 1 min. Discard flow-throughand collection tube.

5. Place the DNeasy spin column in a new 2 mlcollection tube (provided), add 500 µl BufferAW1, and centrifuge at ≥6000 x g (8000 rpm)for 1 min. Discard flow-through and collectiontube.

6. Place the DNeasy spin column in a 2 mlcollection tube (provided), add 500 µl Buffer AW2, and centrifuge for 3 min at fullspeed to dry the DNeasy membrane. Discardflow-through and collection tube.


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 theDNeasy membrane. Incubate at roomtemperature 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 orseparate tubes. Do not use more than 200 µlBuffer AE as this will cause the eluate to comeinto contact with the DNeasy spin column.


Preparation and lysis of other starting materials

The protocols detailed below generally only differfrom the standard protocols (listed above) at thesample preparation stage.

Protocols for animal blood

These protocols can be used for the isolation ofgenomic DNA from animal blood, as well as buffycoat 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 byvortexing.

5. Incubate for 10 min at 70°C.

6. Continue with step 3 of the “DNeasy Protocolfor 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 byvortexing.

5. Incubate for 10 min at 70°C.

6. Continue with step 3 of the “DNeasy Protocolfor Cultured Animal Cells”.

Protocols for fixed tissues

The DNeasy Tissue Kit can be used to isolategenomic DNA from fixed tissues. The length of DNAisolated depends on the age and type of sample, aswell as the method of fixative used, but is usually<650 bp. Use of fixatives such as alcohol andformalin is recommended. Fixatives that cause cross-linking, such as osmic acid, are notrecommended as it can be difficult to obtainamplifiable DNA from tissues fixed with thesereagents.

Lysis times will vary depending on the sample andtype of tissue.

Yields will depend on size and age of the sample.Yields lower than those obtained using fresh orfrozen tissues are to be expected. Therefore elutingin 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 roomtemperature.

4. Remove supernatant by pipetting. Do notremove any of the pellet.

5. Add 1200 µl absolute ethanol to the pellet toremove residual xylene and mix gently byvortexing.

6. Centrifuge at full speed for 5 min at roomtemperature.

7. Carefully remove the ethanol by pipetting. Donot remove any of the pellet.

8. Repeat steps 5–7 once.

9. Incubate the open microcentrifuge tube at 37°Cfor 10–15 min until the ethanol has evaporated.

10. Resuspend the tissue pellet in 180 µl Buffer ATLand continue with the “DNeasy Protocol forAnimal Tissues” from step 2.


Protocol for formalin-fixed tissue

1. Wash tissue sample twice with PBS to removefixative.

2. Discard PBS and continue with the “DNeasyProtocol 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 at7500 rpm (5000 x g). Discard supernatant.

2. Resuspend pellet in 180 µl Buffer ATL.

3. Continue with the ”DNeasy Protocol for AnimalTissues” from step 2.

Protocol for Gram-positive bacteria

1. Harvest cells (max. 2 x 109 cells) in a micro-centrifuge tube. By centrifuging for 10 min at7500 rpm (5000 x g). Discard supernatant.

2. Resuspend pellet in enzymatic lysis buffer (notprovided; 20 mM Tris·Cl, pH 8.0; 2 mM EDTA;1.2% Triton® X-100; 20 mg/ml lysozyme). Addlysozyme to buffer immediately before use.

3. Incubate for at least 30 min at 37°C.

4. Add 25 µl proteinase K and 200 µl Buffer AL.Mix by vortexing. Do not add proteinase K directly to Buffer AL.

5. Incubate at 70°C for 30 min. If required,incubate at 95°C for 15 min to inactivatepathogens. Note that incubation at 95°C can lead to some DNA degradation.

6. Continue with the “DNeasy Protocol for AnimalTissues” from step 4.

Protocol for yeasts

1. Harvest cells (max. 5 x 107 cells) in a micro-centrifuge tube by centrifuging for 10 min at7500 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 andincubate at 30°C for 30 min. Lysis time andyield will vary depending on cell number andyeast species.

3. Pellet the spheroplasts by centrifuging for 10 min at 300 x g.

4. Resuspend the spheroplasts in 180 µl BufferATL.


Protocols for insects

Two protocols exist for the isolation of genomicDNA from insects, either can be used as desired.

Protocol A

1. Grind up to 50 mg insects in liquid nitrogenwith a mortar and pestle, and place powder ina 1.5 ml microcentrifuge tube.

2. Add 180 µl Buffer ATL.


Protocol B

1. Place up to 50 mg insects in a 1.5 ml micro-centrifuge tube.

2. Add 180 µl PBS and homogenize the sampleusing an electric homogenizer or a disposablemicrotube pestle.


User-Developed Protocols

Isolation of genomic DNA from saliva

1. Ensure that the donor animal has not eaten inthe preceding 30 minutes. Collect 1 ml saliva.

2. Add 4 ml PBS (not provided) to the sample andcentrifuge at 1800 x g for 5 min.

3. Carefully decant the supernatant. Resuspend thepellet in 180 µl PBS.

DNeasy spin columns copurify RNA and DNAin parallel when both are present in the sample.RNA may inhibit some downstream reactions,but it does not inhibit PCR. If RNA-free genomicDNA is required, 20 µl of RNase A stocksolution (20 mg/ml) should be added to thesample before the addition of proteinase K.

4. Add 250 µl proteinase K solution and 200 µlBuffer AL to the sample, mix thoroughly byvotexing, and incubate at 70°C for 10 min.Continue with the “DNeasy Protocol forCultured Animal Cells” from step 3.

In order to ensure efficient lysis, it is essentialthat the sample and buffer AL be mixedimmediately and thoroughly.

Isolation of genomic DNA from nails, hair, or birdfeathers*

1. Cut the sample into small pieces, place in a 1.5 ml microcentrifuge tube, and add 200 µlBuffer X1. Incubate at 55°C for at least 1 h untilthe sample is dissolved. Invert the tubeoccasionally to disperse the sample, or placeon a rocking platform.

Buffer X1: 10 mM Tris·Cl; pH 8.0, 10 mMEDTA, 100 mM NaCl, 40 mM DTT, 2% SDS,250 µg/ml proteinase K. Add proteinase K andDTT immediately before use.

2. Add 200 µl Buffer AL and 200 µl ethanol to thesample and mix by vortexing.

3. Pipet the mixture from step 2 into a DNeasyspin 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 mlcollection 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 collectiontube (provided), add 500 µl Buffer AW2, andcentrifuge for 3 min at full speed to dry theDNeasy membrane. Discard flow-though andcollection tube.

6. Elute the DNA in 50–100 µl Buffer AE ordistilled water.

Elution in 50 µl will yield more concentratedDNA, whereas elution in 100 µl will recover agreater amount of DNA. If the expected amountof DNA is not known, it is better to elute inseveral aliquots of 50 µl, as these can becombined if necessary. Elution of the DNA inBuffer AE is recommended if the DNA is to bestored, since DNA stored in water is subject toacid hydrolysis.

* Feather quills will remain undissolved during step 1, therefore it will benecessary to transfer the supernatant to a new microcentrifuge tube atthe end of step 1.


Isolation of DNA from compact bone

1. Completely remove bone marrow and softtissues using razor blades and/or sandpaper.

2. Crush the bone into small fragments. Grind to afine powder using a mixer mill or a metalblender half-filled with liquid nitrogen.

3. Transfer 5 g powder into sterile 50 ml polypropylene tubes and add 40 ml of 0.5 MEDTA, pH 7.5, to decalcify the sample. Agitatethe 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 thedecalcification process several times.Generally, decalcification takes 3–5 days. Thedecalcification process can be monitored byadding a saturated solution of ammoniumoxalate, pH 3.0, to the decanted supernatant. Ifthe solution remains clear, the decalcificationprocess can be stopped.

5. Wash the pellet with 40 ml sterile deionizedwater to remove ions that have accumulatedduring the decalcification. Centrifuge thesample at 2000 x g for 15 min and discard the supernatant. Repeat this washing procedure3 times.

6. To 50 mg of pellet, add 360 µl Buffer ATL.

7. Add 40 µl proteinase K, mix by vortexing, andincubate at 55°C until the pellet is completelylysed. Vortex occasionally during incubation todisperse the sample, or place in a shakingwater bath or on a rocking platform.

8. Vortex for 15 s. Add 400 µl Buffer AL to thesample, mix thoroughly by vortexing, andincubate at 70°C for 10 min.

9. Add 400 µl ethanol (96–100%) to the sampleand mix thoroughly by vortexing.

10. Pipet up to 650 µl of the mixture from step 9into the DNeasy column placed in a 2 mlcollection tube (provided). Centrifuge at ≥6000 x g. Discard flow-through and collectiontube. Repeat until all of the sample has beenloaded. Continue with the “DNeasy Protocol forAnimal Tissues” from step 6.


References1. Hirsch, H.H. and Bossart, W. (1999)

Two-centre study comparing DNA preparationand PCR amplification protocols for herpessimplex virus detection in cerebrospinal fluidsof patients with suspected herpes simplexencephalitis. J. Med. Virol. 57, 31.

2. Miller, S.A., Dykes, D.D., and Polesky, H.F.(1988) A simple salting-out procedure forextracting 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 MolecularBiology, John Wiley & Sons, New York.

4. Sambrook, J. and Russell, D. (2001) MolecularCloning — A Laboratory Manual, 3rd Ed.,Cold Spring Harbor Laboratory Press, NewYork.

5. Heermann, K.H., Gerlich, W.H., Chudy, M.,Schaefer S., and Thomssen, R. (1999)Quantitative detection of hepatitis B virus DNAin two international reference plasmapreparations. Eurohep Pathobiology Group. J. Clin. Microbiol. 37, 68.

6. Verhagen, O.J., Wijkhuijs, A.J., van der Sluijs-Gelling, A.J., Szczepanski, T., van derLinden-Schrever, B.E., Pongers-Willemse, M.J.,van Wering, E.R., van Dongen, J.J., and vander Schoot, C.E. (1999) Suitable DNA isolationmethod for the detection of minimal residualdisease by PCR techniques. Leukemia 13,1298.

7. Reimann, U., Guntermann, D., and Weber, O.(1998) High-throughput DNA purification withDNeasy 96 — more than just mouse tails. QIAGEN News 1998 No. 3, 7.

8. Schwarz, H. (1997) Rapid high-throughputpurification of genomic DNA from mouse andrat tails for use in transgenic testing. TechnicalTips 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 causesmuscle weakness and atrophy in juvenile andadult mice. Proc. Natl. Acad. Sci. USA 93,13286.

10. Watson, A.J., Fuller, L.J., Jeenes, D.J., andArcher, D.B. (1999) Homologs of aflatoxinbiosynthesis genes and sequence of aflR inAspergillus 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 murinecolonic hyperplasia, exhibits clonality:synonymy of C. rodentium and mouse-pathogenic E. coli. J. Clin. Microbiol. 38, 4343.

12. Maeda, N., Palmarini, M., Murgia, C., andFan, H. (2001) Direct transformation of rodentfibroblasts 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 formultiresistant integrons. Proc. Natl. Acad. Sci.USA 98, 652.


Ordering InformationProduct 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, 69504Buffers, Collection Tubes (2 ml)

DNeasy Tissue Kit (250) 250 DNeasy Spin Columns, Proteinase K, 69506Buffers, 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, 69581Proteinase 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, 69582Proteinase 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



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

Mixer Mill MM 300, Universal laboratory mixer mill 85110100-115V/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 69999with 1.5 or 2.0 ml microcentrifuge tubes on the Mixer Mill MM 300

Tungsten Carbide Beads, Tungsten Carbide Beads, suitable for use 699973 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).


Ordering InformationProduct 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, 81031for use with QIAGEN centrifuges

Accessories

Genomic DNA Buffer Set† Buffers including specific lysis buffers for yeast, 19060bacteria, 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), 1200072304 in packs of 96, and nonsterile polypropylene caps for Collection Microtubes

Elution Microtubes RS Nonsterile polypropylene tubes (0.6 ml); 1200082304 in racks of 96. Includes caps

* The Plate Rotor 2 x 96 is available exclusively from QIAGEN. † Enzymes must be purchased separately.

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Argentina Tecnolab S.A. (011) 4555 0010 Austria/Slovenia VWR International GmbH (01) 576 00 0 Belgium/Luxemburg Westburg b.v.0800-1-9815 Brazil Uniscience do Brasil 011 3622 2320 China Gene Company Limited (852)2896-6283 Cyprus Scientronics Ltd(02) 765 416 Czech Republic BIO-CONSULT spol. s.r.o. (420) 2 417 29 792 Denmark VWR International ApS 43 86 87 88 Egypt Clinilab52 57 212 Finland VWR International Oy (09) 804 551 Greece BioAnalytica S.A. (10)-640 03 18 India Genetix (011)-542 1714or (011)-515 9346 Israel Westburg (Israel) Ltd. 08 6650813/4 or 1-800 20 22 20 Korea LRS Laboratories, Inc. (02) 924-86 97Malaysia RESEARCH BIOLABS SDN. BHD. (603)-8070 3101 Mexico Quimica Valaner S.A. de C.V. (55) 55 25 57 25 The NetherlandsWestburg b.v. (033)-4950094 New Zealand Biolab Scientific Ltd. (09) 980 6700 or 0800 933 966 Norway VWR International AS22 90 00 00 Poland Syngen Biotech Sp.z.o.o. (071) 351 41 06 or 0601 70 60 07 Portugal IZASA PORTUGAL, LDA (21) 424 7312Singapore Research Biolabs Pte Ltd 2731066 Slovak Republic BIO-CONSULT Slovakia spol. s.r.o. (02) 5022 1336 South Africa SouthernCross Biotechnology (Pty) Ltd (021) 671 5166 Spain IZASA, S.A. (93) 902.20.30.90 Sweden VWR International AB (08) 621 34 00Taiwan TAIGEN Bioscience Corporation (02) 2880 2913 Thailand Theera Trading Co. Ltd. (02) 412-5672 In other countries contact:QIAGEN, Germany

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