in vitro transcription--no transcript produced - (Jul/05/2006 )

HI,

i am doing in vitro transcription these days, but it seems no transcripts was produced. I first

linearized the template at the 3' end ,then perform the in vitro transcription .By the way,The

transcript i want is about 10kb I am confused now and don't know how to improve.Any

suggestion will do help me.

DETAILS!!! What template, does it have a promoter (I hope so)? If it does then which one? What RNA polymerase system are you using? What conditions are you carrying your IVT under?

I perform IVT on an albeit shorter fragment (266 bp) every day. This is coupled to a T7 promoter and I use a Promega T7 RNA polymerase with buffer. Works every time. Has never failed.

Details and then maybe we can help

i noticed in couple of exp that the fact of linearized template could help.