In vitro Transcription - Do I need to linearize my plasmid ? (Apr/01/2008 )

Hi, all.


I'm going to perform in vitro transcription. My target is inserted in a plasmid, pET-17b, which has T7 promoter and T7 terminator.

At first, I think T7 terminator would stop the RNA synthesis. But, I'm wonder why all in vitro transcripton kits (as I can find) doesn't contain T7 terminator, and have to make a plasmid to be linearized before performing in vitro transcription.

So, I worry about the efficiency of T7 terminator. I don't know that whether T7 terminator can stop all the transcribing RNA of T7 RNA polymerase.

My question is "Do I need to linearize my plasmid before using for in vitro Transcription ?"

By the way, if you have any suggestion, please let me know.


Thanks, in advance.

Hi,

yes its important to linearize ur plasmid before transcription. Chose a restriction enzyme which is not going to affect ur fulllength transript. It works...by the way whats the size of ur transcription product

Thanks