T7/SP6 riboprobe transcription

Arnoud van Vliet avvliet at knoware.nl
Mon Nov 27 15:50:31 EST 2000


> 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.

Hi Jonathan,

maybe not answering the question exactly, but hopefully useful anyway: we
make our antisense probes using a normal forward primer, and a reverse
primer which has a T7 promoter attached at the 5' end. The sequence we use
for that is:

5'-CTAATACGACTCACTATAGGGAGA

This seems to be enough to give efficient in vitro transcription, as we get
good probes this way. Check the Ambion site (no affiliations) at
http://www.ambion.com/techlib/tb/ for a lot of useful tips for all kinds of
RNA work in their technical bulletins.

hope this helps
Arnoud







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