It's been more than 2 weeks that i'm trying to have a full length cDNA copy of Coxsackievirus but have only fragments. I tried to use oligo dT and specific primers on the 3'End of the viral genome, then I tried to expend the RT (Superscript II, life tech) time to 2 hours instead of 1, I increased the template RNA (i used synthesized full genome RNA, and extraction product of the viral strain) quantity for the reaction, I treated the RT product with Rnase H for 30 min (recommended in case of full length PCR). But I got nothing when trying to amplify the genome with a long PCR (KAppa HiFi, clinisciences). I have tried PCRs on same RT product on the 5' end that works, as well as on the 3', but that does not confirm that the RT worked because our virus can self prim so without any addition of RT primer. It's getting quite depressing. Does anybody have other ideas to try? Thanks
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