Hello! I am having difficulty amplifying the coding sequence of RSV F protein.

The protocol I have used is a sfollows: I infect HEp-2 cells with the virus, then when CPE is evident I collect the cells and use the RNeasy kit from Qiagen to purify the RNA with good yields. Then I have tried to do the RT with supercript IV or Protoscript (NEB), using gene specific primers, oligodT or random primers. And finally I have tried PCR with different concentrations of template, DMSO, Mg2+, temperatures.

Does anyone have any piece of advise?

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