Download DNA Viruses: Methods and Protocols by Paul M. Lieberman PDF

By Paul M. Lieberman

A compendium of quite simply reproducible and novel ways to manage DNA viruses and represent their assorted organic homes. The authors emphasize ideas for viral detection and genetics, but additionally comprise tools for constitution selection, gene expression, replication, pathogenesis, advanced mobile types, recombinant genetics, and computational/systems ways. Wide-ranging and hugely useful, DNA Viruses: equipment and Protocols will stimulate new instructions in virology learn with its novel ideas for engineering viral vectors in gene remedy, and its complex ways for detecting viruses in human sickness.

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The cellular housekeeping gene U1A snRNP (primers U1A1 and U1A2). 2 µL AmpliTaq DNA polymerase. Multiprimed EBV RT-PCR 33 4. Cycle the samples in a PCR device using the following PCR program: 4 min at 95°C; 40 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C; 7 min at 72°C, and finally a hold at 4°C (see Note 15). 4. 1. Electrophoretic Separation of PCR Products and Blotting to Nylon 1. 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.

For this we prefer the NucliSens® Nucleic Acid Isolation Method (BioMerieux, Boxtel, The Netherlands), which is a silica-based RNA extraction method efficiently removing substances putatively interfering with amplification in RT-PCR (10,11). Make sure that no organic phase is removed. It is advised to leave a small amount of aqueous phase on top to avoid this. The RNA/isopropanol solution can be stored long term at –80°C. Always keep on ice when pipeting the required volume from the RNA/isopropanol stock solution and return solution to –80°C immediately after use.

42 3. 4. 5. 6. Hochberg and Thorley-Lawson Heparinized blood sample. Ficoll-Hypaque (Pharmacia). 5% bovine serum albumin/1X phosphate-buffered saline. 5 M Acetic acid. 2. Quantitative Determination of the Frequency of Virus-Infected Cells: DNA-PCR 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 10 mg/mL Proteinase K. 3. 5% Tween-20. 5% NP-40. 96-Well V-bottom plate (Falcon). 55°C Incubator. dNTP mix (10 mM each dNTP; Invitrogen). Diethyl pyrocarbonate (DEPC) or high-performance liquid chromatography (HPLC) treated water.

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