Genomic DNA Purification Technical Hints, Applications, and Protocols

Genomic DNA Purification
Technical hints, applications, and protocols
Trademarks and disclaimers Patented or patent-pending and/or registered or registration-pending 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.
Contents
Introduction Effects of contaminants on downstream applications Starting materials Sample collection and storage before isolation of genomic DNA Animal tissue Animal, yeast, and bacterial cell cultures Animal blood Amount of starting material DNA yields Disruption and lysis Disruption methods Enzymatic lysis of animal tissue Enzymatic lysis of bacteria and yeast Disruption using rotor-stator homogenizers Disruption using the Mixer Mill MM 300 and other bead mills Disruption using a mortar and pestle DNA isolation methods Preparation of crude lysates Salting-out methods Organic extraction methods Cesium chloride density gradients Anion-exchange methods Silica-based methods DNeasy Tissue Kits Applications using DNeasy Tissue Kits Functional genomics Molecular breeding Genetic studies in bacteria and yeast Disease research Natural history applications Phylogeny and biodiversity studies Detection of viral pathogens Protocols User-Developed Protocols References Ordering Information 4 4 5 6 6 6 7 7 7 8 8 8 8 8 9 9 10 10 11 11 11 12 12 14 14 16 17 18 19 20 21 22 27 29 30
DNA Contamination Leads to Lower Yields and Quality of PCR Products

DN ea sy DN ea sy
Introduction
Over the last decade there has been an increased requirement for isolation of pure genomic DNA that performs well in any downstream 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 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 acids. This guide gives an overview of the techniques used for isolation 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.
Phenol 0.2 0.5%
SDS 0.005 0.01%
DN ea sy
NaCl 25 50 mM
DN ea sy
Ethanol 1 5%
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.
Starting materials
Genomic DNA can be isolated from a wide variety of starting materials, 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
Effect of Contamination on Spectrophotometry

0.4 DNA + phenol (P) Absorbance
DNA (Q)
0.0 220 Wavelength (nm) 320
Figure 2. Effect of phenol on DNA quantification. UV scan of Q (QIAGEN): NA purified using silica-gelmembrane technology (100 l sample); P (phenol): The same sample with 1 l phenol diluted 1:1000 in water (final dilution 1:100,000). Phenol contamination of DNA can interfere with spectrophotometric readings and lead to overestimation.
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.
Table 1. Maximum recommended amounts of starting material*

Animal tissue Mouse tail Rat tail Cultured cells Bacteria Yeast
* Using DNeasy Tissue procedures.
Sample collection and storage before isolation of genomic DNA The quality of the starting material affects the quality and yield of the isolated DNA. Optimal results are obtained with fresh material, or with material that has been immediately frozen (frozen in liquid nitrogen or in a mixture of ethanol and dry ice) and stored at 20C or 70C. Repeated freezing and thawing of stored samples should be avoided, as this leads to reduced fragment size and precipitation of the DNA, and in clinical samples, to reduced yields of pathogen 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 28C or 20C 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 20C, 70C, 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 in shorter fragments upon purification. Fixatives that cause crosslinking, such as osmic acid, are not recommended if DNA will be isolated from the tissue. It is also possible to isolate DNA from paraffin-embedded tissue. Animal, yeast, and bacterial cell cultures Cell cultures are usually harvested by centrifugation followed by removal of the supernatant. Cells are then stored at 20C or 70C. Alternatively, animal cell nuclei can be prepared and stored at 20C. For certain bacterial and yeast cultures that accumulate large amounts of metabolites and/or form very dense cell walls, cells should be harvested in the early log phase of growth, and washed with fresh medium. Fresh or frozen cell pellets can be used.
25 mg 0.61.2 cm 0.6 cm 5 x 106 cells 2 x 109 cells 5 x 107 cells
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 Base pairs per haploid genome 4.6 x 106 1.2 x 107 5.4 x 107 1.2 x 108 9.7 x 107 1.8 x 108 1.2 x 109 3.0 x 109 3.0 x 109 3.1 x 109 3.3 x 109
Animal blood Freshly drawn animal whole blood (nucleated or non-nucleated) should be digested with proteinase K. The amount of blood used is critical and tenfold less nucleated blood should be used in the procedure compared with non-nucleated blood. Neither type of blood should be coagulated. Buffer AL should be added to blood containing proteinase K and incubated for 10 minutes at 70C for digestion. The appropriate DNeasy Tissue Kit protocol should then be followed. Amount of starting material The amount of starting material to use is a key point in the successful isolation of genomic DNA. Starting sample size depends on the tissue or cells being used, but the amounts shown in Table 1 (page 6) are a good guide when using DNeasy Tissue procedures. Often, an excess of starting material can be counter-productive, and as a general guide less is more. If you have no information on DNA content and your starting material is not shown in Table 1, we recommend beginning with half the maximum amount of starting material indicated in Table 1. Depending on the yield obtained, the sample size can be increased in subsequent preparations. DNA yields 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.
Table 3. Yields of genomic DNA with DNeasy Tissue Kits

Yield Source Lymphocytes (5 x 106) HeLa cells (2 x 106) Liver (25 mg) Brain (25 mg) Lung (25 mg) Heart (25 mg) Kidney (25 mg) Spleen (10 mg) Mouse tail, 1.2 cm (tip section) Rat tail, 0.6 cm (tip section)
* Nucleic acids purified without RNase treatment. Nucleic acids purified with RNase treatment.
Total nucleic acids (g)* 2030 4060 60115 3560 820 2545 4085 2545 1530 2560
DNA (g) 1525 1525 1030 1530 510 510 1530 530 1025 2040
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 590 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 l 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.
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 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 (diameter) 37 mm 0.5 mm 0.1 mm 0.5 mm
Efficient High-Throughput Disruption of Tissue Samples
Starting sample Animal tissues Animal cells Bacteria Yeast
Bead type Stainless steel Glass Glass Glass
Figure 3. The Mixer Mill MM 300 allows processing of up to 192 tissue samples in just 24 minutes.
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-nitrogencooled, 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.
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. 2. 3. 4. Disruption Lysis Removal of proteins and contaminants 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., 90C 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.
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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.
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DNeasy Tissue Spin and 96-Well Plate Procedures

Tissue sample Mouse tail or animal tissue
Anion-exchange methods Solid-phase anion-exchange chromatography is based on the 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 RNA, cellular proteins, and metabolites are washed away using medium-salt buffers, and high-quality DNA is eluted using a high-salt 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 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 50100 kb. QIAGEN offers QIAGEN Genomic-tips for the purification of high-molecularweight DNA. Silica-based methods DNeasy Tissue Kits
Lyse
Collect mouse tails or tissue samples and lyse
Bind DNA
Bind DNA
Wash
Wash
Elute
Ready-to-use DNA
Elute into Elution Microtubes RS
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 remain in solution and are subsequently washed away. This is simpler and more effective than other methods where precipitation or extraction is required. Ready-to-use DNA is then eluted from the silica-gel membrane using a low-salt buffer. No alcohol precipitation is required, and resuspension of the DNA, which is often difficult if the DNA has been over-dried, is not required. DNeasy Tissue Kits are designed for rapid isolation of pure total DNA (genomic, viral, and mitochondrial) from a wide variety of sample sources, including fresh and frozen animal cells and tissues, 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 2030 kb. DNA of this length is particularly suitable for PCR analysis as well as Southern blotting analysis (713). 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.
Ready-to-use DNA
Figure 4. The DNeasy Tissue spin and 96-well plate procedures.
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The DNeasy Tissue procedure is suitable for both very small and 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 overload the DNeasy System, as this can lead to significantly lower yields than expected (Figure 5). Overloading the DNeasy System can also adversely affect the purity of the DNA (Figure 6).
DNA yield (g)
Effect of Sample Size on DNA Yield

50 Liver Salivary gland Kidney
40 30
The DNeasy Tissue procedure is also highly suited for purification of DNA from very small amounts of starting material. If the sample has less than 5 ng DNA (<10,000 copies), 35 ng carrier DNA (a homopolymer such as poly dA, poly dT, or gDNA) should be added to the starting material. Ensure that the carrier DNA does not interfere with the downstream application.
20
10
0 10
15
20 Tissue (mg)
25
30
DNA Purity and Time Required for Different Isolation Methods
High QIAGEN
DNeasy Tissue Kits
QIAGEN
Anionexchange technology
1 x CsCl gradient
Figure 5. DNA was purified from tissue samples pooled from 10 mice using the DNeasy Tissue protocol for rodent tails. DNA yield was determined spectrophotometrically. Each point represents the mean and standard deviation from 10 preparations.
Purity
Organic extraction Saltingout
Effect of Sample Size on DNA Purity

2.1
Low Less
A260/A280 ratio
Simple methods*
alcohol Alcohol precipiprecipitation tation
Liver Salivary gland Kidney
2.0
Time
More
1.9
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 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, 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-salt buffer. This is incubated and the proteins precipitated by centrifugation. The supernatant is then subject to alcohol precipitation. See pages 1012 for further information on DNA isolation methods. * No proteinase K digestion.
1.8
1.7 10 15 20 Tissue (mg) 25 30
Figure 6. The DNA purity of the samples described in Figure 5 was determined spectrophotometrically by measuring the A260/A280 ratio.
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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

+/+
ne o1 ne o2
/
ne o1 ne o2
w t
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
w t
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. Baxhomozygous 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 glutamateinduced nerve cell death. Journal of Neurochemistry 76, 295. Published with permission.
Genotyping of Transgenic Tadpoles by PCR

N oc c G on FP tro co l nt ro l
GFP Noc 1 2 3 4 5 6 7
500 bp 250 bp
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 27), 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.
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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
parent
Allele size ranges JOE/L
JOE/S -36 JOE/S -42 TAMRA -31 FAM27
JOE/18
TAMRA FAM
parent
JOE/S
Offspring
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 510 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 l elution buffer and 1 l was used for multiplex 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 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

kb 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.6
5A 3 AT CC B3 13 14 87 14 88 14 89 14 90 14 91 14 92 14 93 14 94 14 95 14 96 14 97 14 98 14 99 15 00 15 01 15 02 15 03
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 Zckert, 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.
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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

Without PH
0. 5 50 5
0. 05 0. 00 5 N o DN A
With PH
50 5
0. 5 Co nt ro l 50 DN A 5
Normal
0. 5
ng DNA 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

Copies of tumor-specific translocation
7 70 700 7000
Figure 14. DNA was purified using the DNeasy Tissue Kit. A chromosomal translocation in the Burkitts 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 Quencher1 (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.
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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 Washed Dry bone A Unwashed Long digestion Washed Unwashed
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 tissue 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 positive 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.
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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 worlds 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
100 100 99 59
85
M. ravelobensis M. tavaratra M. myoxinus
66 100
M. berthae
63
Figure 16. The mouse lemur, which is native to Madagascar. (Photograph courtesy of David Haring, Duke University Primate Center, Durham, NC, USA).
99
M. rufus1
98
100 100 98
M. rufus2 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.
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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
PC R: 0 dp nP C R: i PC 0 R: dpi 4 nP dp C R: i PC 4 R: dp i nP 28 dp C R: i nP 28 C R: dpi nP 0 dp C R i nP : 5 dp C R: i nP 21 C d R: p ne 47 i ga dp ti PC ve i c on R: nP PC trol C V2 se R: m en PC V2
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., Mller, 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.
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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.
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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 microcentrifuge tube, and add 180 l Buffer ATL. It is advisable to cut the tissue into small pieces for efficient lysis. Add 20 l proteinase K, mix by vortexing, and incubate at 55C 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 13 h, though samples can be lysed overnight. Optional: RNase treatment of the sample. Add 4 l 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 l Buffer AL to the sample, mix thoroughly by vortexing, and incubate at 70C for 10 min. Add 200 l ethanol (98100%) 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. Place the DNeasy spin column in a new 2 ml collection tube (provided), add 500 l Buffer AW1, and centrifuge at 6000 x g (8000 rpm) for 1 min. Discard flow-through and collection tube. Place the DNeasy spin column in a new 2 ml collection tube (provided), add 500 l Buffer AW2, and centrifuge for 3 min at full 2. 9. 8.
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. Place the DNeasy spin column in a clean 1.5 ml or 2 ml collection tube (not provided), and pipet 200 l 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. Repeat elution once as described in step 8. Elution can be performed in the same or separate tubes. Do not use more than 200 l Buffer AE as this will cause the eluate to come into contact with the DNeasy spin column.
2.
DNeasy protocol for rodent tails 1. Cut one (rat) or up to two (mouse) 0.40.6 cm lengths of tail into a 1.5 ml microcentrifuge tube. Add 180 l 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 0.40.6 cm. Add 20 l proteinase K, mix by vortexing, and incubate at 55C 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 68 h, but samples can be lysed overnight. Optional: RNase treatment of the sample. Add 4 l 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.
4.
6.
7.
23
3.
Vortex for 15 s. Add 400 l Buffer ALethanol 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.
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 l RNase A (100 mg/ml) and incubate for 2 min at room temperature. 2. Add 20 l proteinase K and 200 l Buffer AL to the sample, mix thoroughly by vortexing, and incubate at 70C for 10 min. Do not add proteinase K directly to Buffer AL. Add 200 l ethanol (96100%) 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. 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. Place the DNeasy spin column in a new 2 ml collection tube (provided), add 500 l Buffer AW1, and centrifuge at 6000 x g (8000 rpm) for 1 min. Discard flow-through and collection tube. Place the DNeasy spin column in a 2 ml collection tube (provided), add 500 l 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 l 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. Repeat elution once as described in step 7. Elution can be performed in the same or separate tubes. Do not use more than 200 l Buffer AE as this will cause the eluate to come into contact with 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. Place the DNeasy spin column in a new 2 ml collection tube (provided), add 500 l Buffer AW1, and centrifuge at 6000 x g (8000 rpm) for 1 min. Discard flow-through and collection tube. Place the DNeasy spin column in a new 2 ml collection tube (provided), add 500 l 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.
3.
5.
4.
6.
5.
7.
Place the DNeasy spin column in a clean 1.5 ml or 2 ml collection tube (not provided), and pipet 200 l 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. Repeat elution once as described in step 7. Elution can be performed in the same or separate tubes. Do not use more than 200 l Buffer AE as this will cause the eluate to come into contact with the DNeasy spin column.
6.
8.
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).
8.
24
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 l proteinase K into the bottom of a 1.5 ml microcentrifuge tube (not provided). Add 50100 l anticoagulated blood. Adjust the volume to 220 l with PBS. Add 200 l Buffer AL. Mix thoroughly by vortexing. Incubate for 10 min at 70C. Continue with step 3 of the DNeasy Protocol for Cultured Animal Cells.
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 50100 l 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. Add 1200 l xylene. Vortex vigorously. Centrifuge at full speed for 5 min at room temperature. Remove supernatant by pipetting. Do not remove any of the pellet. Add 1200 l absolute ethanol to the pellet to remove residual xylene and mix gently by vortexing. Centrifuge at full speed for 5 min at room temperature. Carefully remove the ethanol by pipetting. Do not remove any of the pellet. Repeat steps 57 once. Incubate the open microcentrifuge tube at 37C for 1015 min until the ethanol has evaporated.
2. 3. 4.
5. 6.
2. 3.
Protocol for whole nucleated blood For use with chicken and goldfish blood. 1. Pipet 20 l proteinase K into the bottom of a 1.5 ml microcentrifuge tube (not provided). Add 510 l anticoagulated blood. Adjust the volume to 220 l with PBS. Add 200 l Buffer AL. Mix thoroughly by vortexing. Incubate for 10 min at 70C. Continue with step 3 of the DNeasy Protocol for Cultured Animal Cells.
4.
5.
2. 3. 4.
6.
7.
5. 6.
8. 9.
10. Resuspend the tissue pellet in 180 l 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. Discard PBS and continue with the DNeasy Protocol for Animal Tissues.
Protocol for yeasts 1. Harvest cells (max. 5 x 107 cells) in a microcentrifuge tube by centrifuging for 10 min at 7500 rpm (5000 x g). Discard supernatant. Resuspend the pellet in 600 l sorbitol buffer (1 M sorbitol; 100 mM EDTA; 14 mM -mercaptoethanol). Add 200 units lyticase and incubate at 30C for 30 min. Lysis time and yield will vary depending on cell number and yeast species. Pellet the spheroplasts by centrifuging for 10 min at 300 x g. Resuspend the spheroplasts in 180 l Buffer ATL. Continue with the DNeasy Protocol for Animal Tissues from step 2.
2.
2.
Protocols for bacteria Protocol for Gram-negative bacteria 1. Harvest cells (max. 2 x 109 cells) in a microcentrifuge tube by centrifuging for 10 min at 7500 rpm (5000 x g). Discard supernatant. Resuspend pellet in 180 l Buffer ATL. Continue with the DNeasy Protocol for Animal Tissues from step 2. 5. 3.
4.
2. 3.
Protocol for Gram-positive bacteria 1. Harvest cells (max. 2 x 109 cells) in a microcentrifuge tube. By centrifuging for 10 min at 7500 rpm (5000 x g). Discard supernatant. Resuspend pellet in enzymatic lysis buffer (not provided; 20 mM TrisCl, pH 8.0; 2 mM EDTA; 1.2% Triton X-100; 20 mg/ml lysozyme). Add lysozyme to buffer immediately before use. Incubate for at least 30 min at 37C. Add 25 l proteinase K and 200 l Buffer AL. Mix by vortexing. Do not add proteinase K directly to Buffer AL. Incubate at 70C for 30 min. If required, incubate at 95C for 15 min to inactivate pathogens. Note that incubation at 95C can lead to some DNA degradation. Continue with the DNeasy Protocol for Animal Tissues from step 4. 2. 3. Protocols for insects Two protocols exist for the isolation of genomic DNA from insects, either can be used as desired. Protocol A 1. Grind up to 50 mg insects in liquid nitrogen with a mortar and pestle, and place powder in a 1.5 ml microcentrifuge tube. Add 180 l Buffer ATL. Continue with the DNeasy Protocol for Animal Tissues from step 2.
2.
3. 4.
5.
Protocol B 1. Place up to 50 mg insects in a 1.5 ml microcentrifuge tube. Add 180 l PBS and homogenize the sample using an electric homogenizer or a disposable microtube pestle.
6.
2.
26
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. Add 4 ml PBS (not provided) to the sample and centrifuge at 1800 x g for 5 min. Carefully decant the supernatant. Resuspend the pellet in 180 l 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 l of RNase A stock solution (20 mg/ml) should be added to the sample before the addition of proteinase K. 4. Add 250 l proteinase K solution and 200 l Buffer AL to the sample, mix thoroughly by votexing, and incubate at 70C 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.
2.
Add 200 l Buffer AL and 200 l ethanol to the sample and mix by vortexing. 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. Place the DNeasy column in a new 2 ml collection tube (provided), add 500 l Buffer AW1, and centrifuge for 1 min at 6000 x g for 1 min. Discard flow-through and collection tube. Place the DNeasy column in a 2 ml collection tube (provided), add 500 l Buffer AW2, and centrifuge for 3 min at full speed to dry the DNeasy membrane. Discard flow-though and collection tube. Elute the DNA in 50100 l Buffer AE or distilled water. Elution in 50 l will yield more concentrated DNA, whereas elution in 100 l 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 l, 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.
3.
2.
4.
3.
5.
6.
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 l Buffer X1. Incubate at 55C 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 TrisCl; pH 8.0, 10 mM EDTA, 100 mM NaCl, 40 mM DTT, 2% SDS, 250 g/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.
27
Isolation of DNA from compact bone 1. Completely remove bone marrow and soft tissues using razor blades and/or sandpaper. Crush the bone into small fragments. Grind to a fine powder using a mixer mill or a metal blender half-filled with liquid nitrogen. 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 4C for 24 h. Centrifuge the sample at 2000 x g for 15 min. Discard the supernatant. Repeat the decalcification process several times. Generally, decalcification takes 35 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. 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.
6. 7.
To 50 mg of pellet, add 360 l Buffer ATL. Add 40 l proteinase K, mix by vortexing, and incubate at 55C 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.
2.
3.
8. Vortex for 15 s. Add 400 l Buffer AL to the sample, mix thoroughly by vortexing, and incubate at 70C for 10 min. 9. Add 400 l ethanol (96100%) to the sample and mix thoroughly by vortexing.
4.
10. Pipet up to 650 l 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.
28
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. 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. 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. Sambrook, J. and Russell, D. (2001) Molecular Cloning A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, New York. 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. 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. 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. 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. 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.
9.
2.
3.
4.
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 rodentium the causative agent of transmissible murine colonic hyperplasia, exhibits clonality: synonymy of C. rodentium and mousepathogenic 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.
5.
6.
7.
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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, Buffers, Collection Tubes (2 ml) DNeasy Tissue Kit (250) 250 DNeasy Spin Columns, Proteinase K, 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, 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, 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 QIAGEN Genomic-tip 100/G QIAGEN Genomic-tip 500/G 25 columns 25 columns 10 columns 10223 10243 10262 69582 69581 69506 69504
Mixer Mill MM 300 for efficient high-throughput disruption of biological samples Mixer Mill MM 300, 100-115V/50-60Hz Mixer Mill Adapter Set (2 x 96) Universal laboratory mixer mill (100/115 V, 50/60 Hz) 2 Sets of Adapter Plates and 2 racks for use with 1.5 or 2.0 ml microcentrifuge tubes on the Mixer Mill MM 300 Tungsten Carbide Beads, 3 mm (200) Tungsten Carbide Beads, suitable for use with 1.2 ml Collection Microtubes 69997 69999 85110
* 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).
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Ordering Information
Product Contents Cat. No. 96-well plate centrifugation system for high-throughput purification procedures Centrifuge 4-15C (120 V, 60 Hz) Centrifuge 4K15C (220 V, 60 Hz) Plate Rotor 2 x 96* Universal laboratory centrifuge with brushless motor (120 V, 60 Hz) Universal refrigerated laboratory centrifuge with brushless motor (220 V, 60 Hz) Rotor for 2 QIAGEN 96-well plates, for use with QIAGEN centrifuges Accessories Genomic DNA Buffer Set Buffers including specific lysis buffers for yeast, bacteria, cells, blood, and tissue: Y1, B1, B2, C1, G2, QBT, QC, QF; for 75 mini-, 25 midi-, or 10 maxipreps Buffer AW1 (concentrate, 242 ml) Buffer AW2 (concentrate, 324 ml) Buffer AL (216 ml) Buffer ATL (200 ml) Buffer AE (240 ml) QIAGEN Proteinase K (2 ml) QIAGEN Proteinase K (10 ml) Collection Tubes (2 ml) Collection Microtubes and Caps 242 ml Wash Buffer (1) Concentrate for 1000 spin, 250 midi, or 100 maxi preps 324 ml Wash Buffer (2) Concentrate 216 ml for 1000 preps 200 ml Tissue Lysis Buffer for 1000 preps 240 ml Elution Buffer for 1000 preps 2 ml (>600 mAU/ml, solution) 10 ml (>600 mAU/ml, solution) 1000 Collection Tubes (2 ml) Nonsterile polypropylene tubes (1.2 ml), 2304 in packs of 96, and nonsterile polypropylene caps for Collection Microtubes Elution Microtubes RS Nonsterile polypropylene tubes (0.6 ml); 2304 in racks of 96. Includes caps
* The Plate Rotor 2 x 96 is available exclusively from QIAGEN. Enzymes must be purchased separately.
81010
81210
81031
19060
19081
19072 19075 19076 19077 19131 19133 19201 120007
120008
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www.qiagen.com
QIAGEN: Australia Tel. 03-9489-3666 Fax 03-9489-3888 Canada Tel. 800-572-9613 Fax 800-713-5951 Distributors: Argentina Tecnolab S.A. (011) 4555 0010 Austria/Slovenia VWR International GmbH (01) 576 00 0 Belgium/Luxemburg Westburg b.v. France 0800-1-9815 Brazil Uniscience do Brasil 011 3622 2320 China Gene Company Limited (852)2896-6283 Cyprus Scientronics Ltd Tel. 01-60-920-930 Fax 01-60-920-925 (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 Clinilab Japan Tel. 03-5547-0811 Fax 03-5547-0818
Germany Italy Tel. 02103-29-12400 Tel. 02-33430411 Fax 02103-29-22022 Fax 02-33430426 Switzerland Tel. 061-319-30-31 Fax 061-319-30-33
UK and Ireland USA Tel. 01293-422-999 Tel. 800-426-8157 Fax 01293-422-922 Fax 800-718-2056
52 57 212 Finland VWR International Oy (09) 804 551 Greece BioAnalytica S.A. (10)-640 03 18 India Genetix (011)-542 1714 or (011)-515 9346 Israel Westburg (Israel) Ltd. 08 6650813/4 or 1-800 20 22 20 Korea LRS Laboratories, Inc. (02) 924-86 97 Malaysia RESEARCH BIOLABS SDN. BHD. (603)-8070 3101 Mexico Quimica Valaner S.A. de C.V. (55) 55 25 57 25 The Netherlands Westburg b.v. (033)-4950094 New Zealand Biolab Scientific Ltd. (09) 980 6700 or 0800 933 966 Norway VWR International AS 22 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 7312 Singapore Research Biolabs Pte Ltd 2731066 Slovak Republic BIO-CONSULT Slovakia spol. s.r.o. (02) 5022 1336 South Africa Southern Cross Biotechnology (Pty) Ltd (021) 671 5166 Spain IZASA, S.A. (93) 902.20.30.90 Sweden VWR International AB (08) 621 34 00 Taiwan TAIGEN Bioscience Corporation (02) 2880 2913 Thailand Theera Trading Co. Ltd. (02) 412-5672 In other countries contact: QIAGEN, Germany
1019469
05/2002

Genomic DNA Purification Technical Hints, Applications, and Protocols

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Genomic DNA Purification Technical Hints, Applications, and Protocols

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Genomic DNA Purification

Technical hints, applications, and protocols

2002 QIAGEN, all rights reserved.