T7/SP6 riboprobe transcription

[Peter Ashby]

In article <3A22725A.7D496BFF at bbsrc.ac.uk>, Jonathan Howarth 
<jonathan.howarth at bbsrc.ac.uk> wrote:

> Hello all,
> 
> Can anyone tell me whether I can amplify an insert in pUBS with T7 and
> SP6 primers and then use the PCR product for riboprobe transcription
> with T7 and SP6 RNA polymerases?
> 
> I just wondered whether transcription would be inefficient with the
> recognition sequences for the polymerses being right at the ends of the
> fragment.
> 
> Any advice would be greatly appreciated to jonathan.howarth at bbsrc.ac.uk

In my experience the answer is yes, the transcription will be 
inefficient. The details are more complex but basically amplifying with 
M13 in KS and then transcribing worked much better, presumably because 
of the added 5-prime bases. I don't know how many bases you need to add 
to improve transcription sufficiently.

Peter

-- 
Peter Ashby
Wellcome Trust Biocentre
University of Dundee
Dundee, Scotland
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