Protocols and Applications Guide (US Letter Size) - Promega

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||||||||| 9DNA Purification VI. Genomic DNA Isolation Systems Purified genomic DNA is necessary for further analysis of disease states, single nucleotide polymorphisms (SNPs) and for many other multiplex and real-time PCR applications. Many methods exist for isolation of chromosomal DNA, and Promega has genomic purification systems that are both general (able to isolate from many source materials) or specialized (primarily used for one source type). The source types range from bacteria to humans and can encompass tissues from blood to muscle and from leaf to seed. A. Wizard® Genomic DNA Purification Kit The Wizard® Genomic DNA Purification Kit (Cat.# A1120, A1125, A1620) is both a versatile and scalable system for isolating genomic DNA. With this system alone, chromosomal DNA can be isolated from whole blood (Walker et al. 2003), plant leaf (Zhang et al. 2004), Gram-positive (van Schaik et al. 2004) and Gram-negative bacteria (Flashner et al. 2004), mouse tail (Lee et al. 2005) and yeast (Martinez et al. 2004). Additional sample types like fungus (Ahmed et al. 2003), infected frog tissues embedded in paraffin (Pereira et al. 2005), saliva (Cox et al. 2004) and flour beetles (Lorenzen et al. 2002) have also been used successfully with the Wizard® Genomic DNA Purification Kit. Not only is this genomic purification system successful with many sample types, it is also easily scaled for the quantity of starting material by adjusting reagent volumes to accommodate your needs. Additional references for the Wizard® Genomic DNA Purification Kit or any of the Promega DNA isolation systems can be found on our Citations web site (www.promega.com/citations/). Additional Resources for the Wizard® Genomic DNA Purification Kit Technical Bulletins and Manuals TM050 Wizard® Genomic DNA Purification Kit Technical Manual (www.promega.com /tbs/tm050/tm050.html) Promega Publications PN073 Using the Wizard® Genomic DNA Purification Kit with 96-well plates (www.promega.com /pnotes/73/8235_20/8235_20.html) PN068 Isolation of Spirochete DNA using the Wizard® Genomic DNA Purification Kit (www.promega.com /pnotes/68/7381_30/7381_30.html) eNotes Wizard® Genomic DNA Purification Kit provides high-quality genomic DNA template for molecular phylogenetic studies on Copepod crustaceans (www.promega.com /enotes/applications/ap0063.htm) Protocols & Applications Guide www.promega.com rev. 3/09 eNotes Wizard® Genomic DNA Purification Kit and the isolation of plant genomic DNA (www.promega.com /enotes/applications/ap0027_tabs.htm) eNotes Isolation of genomic DNA from small volumes of whole blood using the Wizard® Genomic DNA Purification Kit (www.promega.com /enotes/applications/ap0011_tabs.htm) eNotes Extraction and amplification of DNA from an ancient moss (www.promega.com /enotes/applications/ap0004_tabs.htm) Online Tools Sample Types Processed with the Wizard® Genomic DNA Purification Kit (www.promega.com /applications/dna_rna/sampletypes/wizgensampletypes.htm) Citations El Sheikh, A.F., Poret-Peterson, A.T. and Klotz, M.G. (2008) Characterization of two new genes, amoR and amoD, in the amo operon of the marine ammonia oxidizer Nitrosococcus oceani ATCC 19707. Appl. Environ. Microbiol. 74, 312–8. These authors investigated the amo operon of the marine ammonia oxidizer Nitrosococcus oceani. The bacteria were grown at 30°C for 3 weeks in 200–400ml batch cultures in artificial seawater in the dark without shaking. Genomic DNA was isolated from cells in stationary phase using the Wizard® Genomic DNA Purification Kit. The isolated DNA was then used for PCR analysis. PubMed Number: 17993553 Gomes, R. et al. (2008) Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model. Proc. Natl. Acad. Sci. USA 105, 7845–50. The authors tested the ability of the salivary proteins of sand fly, the Leishmania vector, to protect against cutaneous leishmaniasis in hamsters. Genomic DNA from 300µl of blood and 100mg of spleen and liver tissue of intradermal inoculated (infected) hamsters or control hamsters was isolated using the Wizard® Genomic DNA Purification Kit. PCR determined the parasite burden using primers that amplified a 120bp conserved region of the Leishmania kDNA minicircle. PubMed Number: 18509051 B. Wizard® SV Genomic DNA Purification System The Wizard® SV Genomic DNA Purification System (Cat.# A2360, A2361) provides a fast, simple technique for the preparation of purified and intact DNA from mouse tails, tissues and cultured cells in as little as 20 minutes, depending on the number of samples processed (up to 24 by centrifugation, depending on the rotor size, or up to 20 by vacuum). With some modifications, whole blood can also be used with this isolation system (Promega Corporation, 2002). This is a silica membrane-based system, PROTOCOLS & APPLICATIONS GUIDE 9-18
||||||||| 9DNA Purification meaning there are limitations to the amount of material that can be loaded onto a single SV column; up to 20mg of tissue (mouse tail or animal tissue) or between 1 × 104 and 5 × 106 tissue culture cells can be processed per purification. With more sample, the prepared lysate may need to be split among two or more columns to avoid clogging the column. For the single-column isolation, a vacuum manifold or a microcentrifuge can be used for sample processing. The technology is based on binding of the DNA to silica under high-salt conditions. In the case of the Wizard® SV Genomic DNA Purification System, the silica is present in a membrane format in a small column. The key to isolating any nucleic acid with silica is the presence of a chaotropic salt like guanidine hydrochloride. Chaotropic salt present in high quantities is able to disrupt cells, deactivate nucleases and allow nucleic acid to bind to silica. Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the membrane. Once the washes are finished, the genomic DNA is eluted under low-salt conditions using either nuclease-free water or TE buffer. The genomic DNA isolated with the Wizard® SV Genomic DNA Purification System is of high quality and serves as an excellent template for agarose gel analysis, restriction enzyme digestion and PCR analysis as seen in Figure 9.10. Table 9.3 provides typical yields of genomic DNA purified from a variety of sources. M –C +C Spin Tail Spin Liver Spin Heart Spin Brain Vacuum Tail Vacuum Liver Vacuum Heart Vacuum Brain Figure 9.10. Amplification of genomic DNA isolated from various tissue sources using the Wizard® SV Genomic DNA Purification System. One microliter of purified genomic DNA was amplified using PCR Master Mix (Cat.# M7502) and mouse-specific IL-1β primers (1.2kb product). Reactions with Mouse Genomic DNA (Cat.# G3091; +C) and without DNA (–C) were performed as positive and negative controls, respectively. Thermal cycling conditions were: one cycle of 3 minutes at 95°C; followed by 30 cycles of: 95°C for 30 seconds, 60°C for 1 minute, 70°C for 1 minute and 30 seconds; final extension at 70°C for 7 minutes; 4°C soak. All lanes contained 10µl of reaction product separated on a 1% agarose gel. PCR products were visualized by ethidium bromide staining. “Spin” and “Vacuum” designations indicate the protocol used for genomic DNA isolation. Protocols & Applications Guide www.promega.com rev. 3/09 3710TA04_2A Table 9.3. Typical Genomic DNA Yield From Various Tissues using the Wizard® SV Genomic DNA Purification System. Sample Amount Average Yield Tail Clipping 20mg 20µg Liver 20mg 15µg Heart 20mg 10µg Brain 20mg 6µg CHO cells 1 × 106 5µg NIH/3T3 cells 1 × 106 9µg 293 cells 1 × 106 8µg Researchers have used this simple and rapid system for many additional sample types and applications including mosquitoes (Stump et al. 2005), mammary stem cells followed by STR analysis (Dontu et al. 2003), Bacillus subtilis (Park et al. 2004), Escherichia coli (Teresa Pellicer et al. 2003), the larval form of the Schistosoma mansoni parasite (Smith et al. 2004) and viral DNA from Kaposi’s sarcoma herpes virus-infected BC3 cells (Ohsaki et al. 2004). Additional Resources for the Wizard® SV Genomic DNA Purification System Technical Bulletins and Manuals TB302 Wizard® SV Genomic DNA Purification System Technical Bulletin (www.promega.com/tbs/tb302/tb302.html) Promega Publications PN081 Introducing the Wizard® SV and SV 96 Genomic DNA Purification Systems (www.promega.com /pnotes/81/9939_09/9939_09.html) eNotes A modified Wizard® SV Genomic DNA Purification System protocol to purify genomic DNA from shed reptile skin (www.promega.com /enotes/applications/ap0089.htm) eNotes Isolation of genomic DNA from bacterial cells using the Wizard® SV Genomic DNA Purification System (www.promega.com /enotes/applications/ap0051_tabs.htm) Online Tools Sample Types Processed with the Wizard® SV Genomic DNA Purification System (www.promega.com /applications/dna_rna/sampletypes/wizSVgensampletypes.htm) Citations Mancuso, M. et al. (2008) Oncogenic bystander radiation effects in Patched heterozygous mouse cerebellum Proc. Natl. Acad. Sci. USA 105, 12445–50. To examine radiation-bystander responses in neonatal mouse cerebellum, heterozygous radiosensitive Patched-1 (Ptch1) mice were exposed to either whole body (WB) X-rays or shielded head/rest of body (SH) irradiation. Genomic DNA was isolated from tumors and normal tissue PROTOCOLS & APPLICATIONS GUIDE 9-19
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CONTENTS I. Nucleic Acid Amplificat
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| 1Nucleic Acid Amplification CONTE
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| 1Nucleic Acid Amplification IX. R
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|| 2RNA Interference CONTENTS I. RN
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||| 3Apoptosis I. Introduction A. C
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||||||| 7Cell Signaling CONTENTS I.
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||||||||||||| 13Cloning CONTENTS I.
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