Tnt Sysetem could not work,help.

[Marko Rehn]

Shuichi Oka (imamura at nibh.go.jp) wrote:
: Dear all
: Does anyone have experience with doing Tnt system (T7 Promotor) ? I'm
: currently trying to  do in vitro translation by using the Tnt system (T7
: Promotor) .It  work in very well with  positive control vector in the kit
: ,but not with my construct vector  ( in pT7 blue : Novagene :  This 
: sequense is perfect.).  Comments from the ones have succeded in such system
: trial are welcomed.

Is your ATG-codon and its surroundings natural one ? We have noticed that not
all sequences are translatable in invitro-translation systems. The TnT-extract
mimics the "natural environment" for translation in contrast to fg. baculo-
virus system where the system forces the things go through. If the 5'-end
of your insert-DNA is not suitable for translation "mama-nature" does not
like it.


Marko Rehn

University of Oulu

Finland