in vitro synthesis of RNA using T7 - (Sep/01/2007 )

I want to in vitro transcribe RNA from DNA that I have amplified by PCR. I have tried out the T7 polymerase kit from Invitrogen, but it didn't work as good as I hoped it would.

Does anyone have any experience with this kit and possibly have a protocol that works to share? Or do you have any other kit/protocol to recommend?

My fragment is approx. 2000 bp.

All tips will be highly appreciated:)

-cathrief-

Hi,

I use Roche kit and it works pretty good for me. Actually what is ur problem when u said it was not good? Low yield?

-kuahmk-

QUOTE (kuahmk @ Sep 4 2007, 02:42 AM)

Hi,

I use Roche kit and it works pretty good for me. Actually what is ur problem when u said it was not good? Low yield?

No yield at all, actually. I have ordered the MegaScript kit from Ambion now. I have also run negative controls with water instead of enzyme, it gives the same bands as all the other reactions, indicating that I only have DNA, not RNA in the sample. Have tried both incubation at 37, 42 and 45 degrees C.

-cathrief-