FAQ: Why cloned templates have to be linearized prior to transcription? Why can't I simply transcribe the circular plasmid containing my target sequence?

T7 RNA Polymerase is an extremely processive enzyme, and will continue to transcribe around a circular template multiple times without disassociating, producing a transcript much longer than the plasmid. Not only will the transcript from each promoter contain mostly vector-derived sequence, but the long length makes coherent annealing into a fully double-stranded product very difficult. Linearization at a point downstream from the cloned target sequence, either by restriction digestion or PCR, will produce an RNA transcript with a length defined by the 3´ end of the template (3), and the resulting RNA duplex will contain only the target sequence flanked by a small amount of vector polylinker sequence.