dig-UTP riboprobes

PDE10 at MOLECULAR-BIOLOGY-1.BIOLOGY.CAMBRIDGE.AC.UK PDE10 at MOLECULAR-BIOLOGY-1.BIOLOGY.CAMBRIDGE.AC.UK
Tue May 18 06:00:00 EST 1993


From: BIO1::PDE10
Date: 18-MAY-1993 11:52:46
Description: RE: transcription with dig-UTP                       

Are you using SP6 polymerase? I find that T3 and T7 give much better yields
than SP6 when making 35-S labelled riboprobes. Indeed I have switched to
using pBluescript instead of pGEM type vectors since it has both T3 and T7
promoters. Alternatively having an insert in both orientations in pGEM allows
use of T3/T7 from either direction to get sense and antisense probes. I have
tried SP6 from several different sources including Stratagene, Promega and
Gibco-BRL with none giving more than 10-20% incorporation, while T3 and T7
consistently give over 85% incorporation. Good luck...
 
Frances Hannan (FLH1000 at phx.cam.ac.uk)



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