HomeFAQsWhy cloned templates have to be linearized prior to transcription? Why can't I simply transcribe the circular plasmid containing my target sequence?
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.
You have been idle for more than 20 minutes, for your security you have been logged out. Please sign back in to continue your session.
Sign in to your NEB account
Any items placed in your cart as a guest will be lost upon sign in or leaving the site.