वायरल डीएनए के Nucleocapsids से तैयार
Summary
हम संक्रमित कोशिकाओं से उच्च शुद्धता herpesvirus nucleocapsid डीएनए अलग करने की प्रक्रिया का वर्णन. अंतिम समाधान से कब्जा कर लिया डीएनए उच्च एकाग्रता और पवित्रता है, यह आदर्श उच्च throughput अनुक्रमण, उच्च विश्वस्तता पीसीआर प्रतिक्रियाओं, और transfections के लिए नए वायरल recombinants उत्पादन के लिए उपयुक्त बना.
Abstract
Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 μg or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections.
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 μl of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.
Protocol
Discussion
इस प्रोटोकॉल के भाग मूल BSL4 परिस्थितियों में वायरल डीएनए अलगाव के लिए विकसित किए गए, लेकिन यह गैर BSL स्थितियों के लिए समान रूप से अच्छी तरह से adapts 4 हम आमतौर पर इस प्रोटोकॉल का उपयोग करने के लिए अल्फा herpesviru…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
लेखक ग्रेग स्मिथ, लीज़ा Pomeranz, मैट Lyman, MARLIES Eldridge, Halina Staniszewska Goraczniak, और ठीक ट्यूनिंग इस प्रोटोकॉल में Enquist प्रयोगशाला के सदस्यों के योगदान की सराहना करते हैं.
Materials
| Name of reagent | Company | Catalogue number | Comments |
| Freon (1,1,2-trichloro-1,2,2-trifluoroethane) | Fisher | T178-4 | Check with your institution for guidelines on appropriate disposal of Freon-containing waste, or see Mendez et al. for potential Freon alternatives.9 |
| Phase lock gel tubes, Heavy 15ml capacity | 5 PRIME | 2302850 | Optional |
| Polyallomer ultracentrifuge tubes | Beckman* | 331372* | *Select tubes appropriate for your own ultracentrifuge; these are included as an example only |
| NP-40 / IGEPAL | Sigma | I-3021 | |
| PK-15 cells | ATCC | CCL-33 | |
| Vero cells | ATCC | CCL-81 | |
| PBS | HyClone | SH30028.03 |
Riferimenti
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