I was trying to make a full-length clone of a circular double-stranded DNA virus genome. The genome is 7,753bp in length. A Rolling Circle Amplification (RCA) was applied with a commercial RCA kit (with a virus-specific primer). After RCA, I chose several restriction enzymes to digest the DNA product, however, none of the enzymes gave an expected band. For example, EcoRI is supposed to have no digestion site therefore should have a smear in the lane after digestion, but there were two bands (~1,800bp and ~2,000bp) showing. A geI is supposed to cut only once therefore it should generate a full-length band with 7,753bp in length, however, the fact was that the only band showing was about 2,000bp. All bands we saw were unexplainable.
I'm wondering if anybody has faced the same problem and/or if you have a solution for that.
Appreciate your collaboration in advance.
Thanks for your answer, Andrei Nikonov, but I don't know why I should have many primers covering the whole genome. Would you please explain more because the manual of the kit says using one primer is enough to amplify the whole genome.
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