DNA Viruses: Methods and Protocols by Paul M. Lieberman

By Paul M. Lieberman

A compendium of conveniently reproducible and novel how you can control DNA viruses and signify their different organic homes. The authors emphasize recommendations for viral detection and genetics, but additionally contain equipment for constitution selection, gene expression, replication, pathogenesis, advanced mobile types, recombinant genetics, and computational/systems ways. Wide-ranging and hugely sensible, DNA Viruses: equipment and Protocols will stimulate new instructions in virology learn with its novel options for engineering viral vectors in gene remedy, and its complicated techniques for detecting viruses in human ailment.

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When needed, samples can be analyzed by standard agarose gel electrophoresis after realtime PCR. For this, capillaries (without stoppers) can be placed upside down in an Eppendorf reagent tube and centrifuged in a benchtop centrifuge for 10 s at low speed, to collect the PCR product. 12. Once the number of plasmid DNA copies per µL standard curve DNA stock is calculated (see Note 4), dilute in steps of 10-fold dilutions to the required concentrations. To decrease pipeting errors, pipet volumes of at least 5 µL in all dilution steps.

Ririe, K. , Andrew, R. , David, D. , Gundry, R. , and Balis, U. J. (1997) The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 22, 176–181. 12. 12 Leight, E. R. and Sugden, B. (2000) EBNA-1: a protein pivotal to latent infection by Epstein-Barr virus. Rev. Med. Virol. 10, 83–100. 13. , Guitierrez, M. , et al. (1996) Variation in the sequence of Epstein Barr virus nuclear antigen 1 in normal peripheral blood lymphocytes and in Burkitt’s lymphomas.

5% agarose gel electrophoresis in TBE buffer for 1–2 h at approx 100 mA. 2. Transfer the PCR products from agarose gel to nylon by standard alkaline capillary blotting in blotting buffer. 3. Mark orientation of the samples on the nylon membrane. 4. Neutralize the nylon membrane by washing three times 5 min in 2X SSC. 5. Air-dry the nylon membrane. 6. Incubate the nylon membrane with 50 mL hybridization buffer in a fully sealed plastic tray placed in a water bath at 55°C for at least 15 min (see Note 16).

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