I would like some help with the following topic:
I have a plasmid with my sequence of interest which is a chimeric virus. For the transcription I inserted a T7 promoter site before my insert. Could you tell me if it is necessary to have a T7 terminator at the end of the insert? thanks
Hi Aline,
The one system I have used (to generate HCV genomic RNA), the answer is no. And it the same answer to many other plasmids that are used to generate virus genomic RNA as well. However, the plasmids need to be linearized with a restriction enzyme, whose site has to be located at the 3' end of the virus sequence. By doing this, the RNA you generate by in vitro transcription won't have plasmid sequence in it.
I hope this information is useful.
Jonny
Hi Aline,
I do agree with Jonny. As he suggested, one need to linearize the plasmid with virus genome inserted to get the viral genomic RNA whether in IVT or co-transfection with T7-RNA pol containing plasmid. Generally, the linearization site is put just after the 3'-end as in some viruses these ends are important in regulation of the replication and translation.
Now what will happen if you do not linearize the plasmid and do not have T7-terminator. In this case, theoretically, the T7-RNA pol will keep on the trascription.
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