help

Olivier Grenet grenet_o at a1.tobias.sandoz.com
Tue Jul 2 02:05:41 EST 1996


Hello sir, 

I'm trying to make an in vitro transcription from a 830bp insert
cloned in pKS Bluscript from Stratagene.
I'm using the pT3 riboprobe system of Promega.
I need a full length RNA, but unfortunatly I obtain 2 of them: The
good one and a shorter one.
I assume that there is a terminator, (hairpin formation), in the
insert.
I've plan to clone the insert in another vector, so that I can
use another polymerase...
I tried already to decrease the temperature of the in vitro
transcription reaction down to 250C, (the kit says so...). I tried
also to increase the temperature up to 400C to avoid hairpin
formation, but in the first case I still obtain 2 products, and
in the second one, no product at all.

Can you tell me if you think to something else that can help me
to avoid the new cloning step.
Thank you.
P.S: From the other side of the insert, the pT7 riboprobe system
is working properly.
Please send help to:
GRENET at dstox.pharma.sandoz.ch




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