Nucleic Acid Isolation.indd - Invitrogen

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Choose the Right Product Fig. 10. Principle for isolation of DNA using Dynabeads ® DNA DIRECT TM Universal. Fig. 11. Dynabeads ® DNA DIRECT TM are suitable for isolating pure, high quality DNA down to 10 cells. DNA was isolated from different numbers of leucocytes, and used for PCR amplification of the GAPDH gene. Product Sample Volume Dynabeads ® DNA DIRECT Universal Dynabeads ® DNA DIRECT Blood Dynabeads ® Genomic DNA Blood Table 3: Which product to choose for your DNA isolation. Genomic DNA Isolation High quality DNA is crucial in DNA diagnostics, mutation detection and SNP analysis, pharmacogenomics, tissue typing and many other applications. Biomagnetic separation with Dynabeads ® offers a simple, rapid and easily automated method for isolating high purity DNA from a variety of different starting samples. Main Advantages of Dynabeads ® DNA Isolation: • PCR-ready DNA isolated in down to 10 minutes. • Eliminate contaminating inhibitors. • Highly reproducible results. • Perfect for automation. PCR-ready genomic DNA of high molecular weight can be isolated using Dynabeads ® . The protocols consist of only a few steps of pipetting and magnetic separation, with all operations performed in a single tube. Contaminating PCR inhibitors are eliminated without any centrifugation steps or use of phenol/chloroform. Choosing the Right Product The Dynabeads ® DNA DIRECT Universal kit (Product no. 630.06) is designed for user-friendly isolation of genomic DNA directly from crude material in only 10 minutes. The kit is particularly useful for isolation of DNA from bacteria and cultured cells, and also from clinical specimens and tissues from various species. Only a minute amount of starting material is required. At least 200 ng of PCR-ready DNA is isolated per sample, sufficient for at least 10 PCR reactions. The Dynabeads ® DNA DIRECT Blood kit (Product no. 631.02) supports 100 isolations from 100 µl of blood per sample, sufficient for at least 100 PCR amplifications. The protocol can be modified to allow isolation from 500 µl blood samples, providing enough template DNA for 1,000 PCR amplifications. The Dynabeads ® Genomic DNA Blood kit (Product no. 634.01/02) is designed for isolation of ultra-pure DNA from 200 µl fresh human blood. In the two-step procedure, leucocytes are first isolated from the erythrocytes and serum by both Dynabeads ® CD15 and CD45. DNA from lysed pure leucocytes is then captured onto DNA binding Dynabeads ® . Fresh/frozen blood ≤ 30 µl Cultured cells ≤ 10 4 cells Animal/plant tissue ≤ 100 mg Bacteria ≤ 10 7 -10 8 cells Fresh or frozen blood, bone marrow Fresh blood (stored up to 4 days at 4-25 0 C) 100-500 µl 200 µl 78 Typical Applications PCR PCR, RFLP, SSCP, Southern Blotting Any downstream application Automated protocols TECAN Genesis ® Kingfisher ® Biomek ® 2000 TECAN Genesis ® Kingfisher ® mL, Biomek ® FX www.dynalbiotech.com
A Variety of Sample Types and Downstream Applications DNA from Bacteria Dynabeads ® DNA DIRECT Universal are particularly useful for rapid isolation of bacterial genomic DNA from bacterial cultures, and also for isolating DNA from bacteria in food, clinical or environmental samples. Specific examples include: Listeria monocytogenes in milk, Leptospira interrogans in blood, cyanobacteria in water samples, Neisseria meningitidis, Haemophilus influenza, Streptococcus pneumoniae, Streptococcus agalactiae, Listeria monocytogenes and E. coli in cerebrospinal fluid and blood, (fig. 12) and Mycobacterium tuberculosis in lung and lymph node tissue. For a complete reference list, please visit www.dynalbiotech.com. DNA from Blood DNA isolation kits from Dynal Biotech are suitable for isolation of DNA from blood. Dynabeads ® DNA DIRECT Universal are recommended for the rapid isolation of DNA from small volumes, or even finger-prick samples, of blood. One test will isolate 600 ng - 1 µg DNA from a 30 µl blood sample, sufficient for 30-50 PCR amplifications. The protocol can be fully automated on most liquid handling robots. The Dynabeads ® DNA DIRECT Blood kit isolates DNA from up to 0.5 ml blood samples in less than 15 minutes. One test provides enough template DNA for up to 1,000 PCR amplifications. The protocol is amenable to semiautomation as it includes only one centrifugation step. Dynabeads ® Genomic DNA Blood kit isolates DNA from 200 µl fresh blood and typically yields between 6-9 µg DNA per sample depending on the number of white blood cells in the blood sample. The protocol is ideal for walk-away automation. DNA from Other Clinical Samples Dynabeads ® DNA DIRECT Universal is excellent for the rapid isolation of DNA from small amounts of solid tumours, mouth wash (fig. 13), buccal scrapes, urine, bile and faeces. After DNA isolation, the beads can be added directly to the PCR without prior elution. The excellent sensitivity allows isolation of DNA even from single cells. DNA for Tissue Typing Dynal Biotech HLA Diagnostics offers a complete range of products for tissue typing. For more information see chapter 9 or go to www.dynalbiotech.com. The Dynabeads ® DNA DIRECT Blood kit is ideal for rapid and simple DNA extraction for low-resolution tissue typing with the Dynal Biotech Reli SSO products. The product isolates DNA from up to 0.5 ml blood, and the DNA is sufficient for at least 13 SSO PCR amplifications. www.dynalbiotech.com 79 A Variety of Sample Types Fig. 12: PCR-products from DNA preparations using the Dynabeads ® DNA DIRECT Universal kit, from the following strains: N. meningitidis (lanes 2-3), H. influenzae (lanes 4-5), S. pneumoniae (lanes 6-7), S. agalactiae (lanes 8-9), L. monocytogenes (lanes 10-11) and E. coli (lanes 12-13). Lanes 1 and 16: MW markers; lane 14: negative control; lane 15: positive PCR-control. MW-sizes of all PCR-products were as expected. Courtesy of A. Bäckman, Örebro Medical Center Hospital, Örebro, Sweden. Fig. 13: PCR on DNA isolated from mouth wash specimen with Dynabeads ® DNA DIRECT TM . Lane 1: 100 bp ladder. Lane 3: extraction control (isolation from water). Lane 4: negative PCR control. Lanes 5-12: 157 bp K-ras PCR product from patient samples. Courtesy of M. Longfellow, Molecular Pathology Laboratory, The General Infirmary, Leeds, UK. 6 7 Nucleic Acid Isolation
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Page 5 and 6: Direct mRNA isolation with the Dyna
Page 7 and 8: mRNA for cDNA Libraries Dynabeads
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Page 15 and 16: Isolating RNA and DNA Binding Prote
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