in vitro translation vector - (Feb/22/2006 )
I'm going to express my protein of interest by using in vitro translation kit from promega (TnT quick coupled trancription/translation system). It uses either T7 or SP6 promoters
I have some doubts on the vector, can I just use pGEM-T easy vector for the cDNA cloning and expression? How can I be sure that the translation starts at ATG of the cDNA, not somewhere else?
Thank you very much:)
The TnT systems uses a coupled T7 transcription and rabbit reticulocyte lysate translation system. Your gene of interest is obviously cloned into the multiple cloning site downstream of the T7 promoter so the transcript will start there and end following the poly(A)n signal triggers a poly A tail to be added. Mammalian translation predominantly follows an ATG within a Kosak consensus sequence. (ttp://www.protocol-online.org/forums/index.php?showtopic=2595)
I've used the kit in a final year project expressing a viral protein and it worked well giving a single band. If there are any other translation initiation points in your open reading frame or ORFs in your cDNA you'll see a few discrete bands. If translation was starting randomly you'd expect a ladder.
All the best,
thanks a lot
One more question...
did you use a radiactive or non-radiactive detection method?
I doubt between the TNT Quick Coupled TnT System (Promega) using S35 methionine or Transcend non-radiactive translation detection system.