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Viral DNA synthesis experiment

The viral DNA synthesis experiment determines when viral DNA synthesis begins.

  1. Infect cells, add 3H-thymidine.
  2. Remove aliquots every 2 minutes, hybridize to filters containing cold phage DNA.
  3. Determine when rise in viral DNA occurs.

Handed DNA encoding ORF3 of Phage 1065. How would you determine whther the .8kb mRNA for ORF3 is amplified after viral DNA synthesis? The first step is to determine when DNA synthesis occurs. See previous problem. Then perform Northern blots to determine if the .8kb mRNA appears either before or after viral DNA syntehsis Polyclonal antibody to ORF3. You also have one to ORF5. To determine if ORF3 is made before or after ORF5 in viral life cycle: infect cells; aliquot oevery 2 minutes for Western blot; probe 2 Western blots (one to ORF3 and one to ORF5) and then determine which is made first.

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