By Andrew D. Yurochko, Eng-Shang Huang (auth.), John Sinclair (eds.)
In Cytomegalovirus Protocols (CMV), John Sinclair and a panel of specialist investigators current a accomplished number of mobile and molecular ideas for the research of cytomegalovirus biology and its pathogenetic mechanisms. The methods-all defined in step by step aspect with prepared reproducibility in mind-range from simple virus tradition to complicated molecular research of CMV constitution and serve as. incorporated are equipment for CMV detection utilizing either immunological and organic recommendations, tools for interpreting basic elements of the CMV an infection cycle, and techniques for reading T cellphone reaction to cytomegalovirus an infection within the human host.
complete and cutting-edge, Cytomegalovirus Protocols offers investigators with a robust choice of the foremost equipment which are illuminating not just the fundamental biology of this complicated and exciting human herpesvirus, but in addition its major position within the pathogenesis of human infectious ailments in addition to their emergent therapies.
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Extra info for Cytomegalovirus Protocols
If the mix exhibits a distinct band of the expected size, purify the remaining volume using a preparative agarose gel. 4. Identify the DNA band using a 365-nm UV lamp and cut it out of the gel, reducing the UV exposure time of the DNA as much as possible. 5. Elute the DNA from the gel slice using the Biotrap chamber and further purify it with Elutip D columns according to the manuals supplied by the manufacturer. 6. Digest the purified DNA with restriction endonucleases BamHI and EcoRI for 2 h.
If a significant amount of the GST-protein partitions in the nonbinding fraction, change to Fractogel. This material has better properties for binding, but less performance compared with Sepharose. It has been reported that the solubility and stability of GST proteins can be improved by lowering the temperature of the culture to 20°C (6). In our hands, this was not successful. The major advantages of the GST system are the GSH affinity purification and the low prevalence of anti-GST antibodies in human sera.
Add 1 mL ammonia solution (BDH) and incubate for 1 h at RT. 3. Centrifuge at 9000 rpm in a benchtop microcentrifuge for 3 min and remove the aqueous layer into a fresh tube. 4. Incubate for 18 h at 55°C. 5. Precipitate oligonucleotides on dry ice for 10 min with 1/10 volume potassium acetate and 3 vol absolute ethanol. 6. Precipitate the pellet at 10,000g for 10 min and wash in 80% ethanol. 7. Resuspend the oligonucleotide pellet in 100 μL of nuclease-free water and determine stock concentration by spectrophotometry at 260 nm.
Cytomegalovirus Protocols by Andrew D. Yurochko, Eng-Shang Huang (auth.), John Sinclair (eds.)