RNase protection using PCR products

[Klaus Salger]

HOFSTRA ANGELA 905-822-3520 (Hofstra at aa.wl.com) wrote:
: Hi,
: I'm interested in using PCR products for making riboprobes for RNase protection
: assays.
: I have a technical bulletin from Ambion that give a brief description; but, if
: anyone has a reliable method for introducing a relevant promoter site and
: subsequent transcription I'd love to read about it.  Please respond or post.
: Thanks,  Angela

Hi Angela,
you just need an oligo which contains the T7 promoter at the 5' end and some
specific, complementary bases.

Here's what my primers looked like:

5' T7 promoter                                                             3'
gtaatacgactcactataggg-20bp of complementary sequence of the 3'-end of my gene
+
20bp of  complementary sequence of the 5'-end
-> PCR -> in vitro transcription

Please tell me, if you need more specific informations.

Cheers
  Klaus

--
Klaus Salger                phone : +49 (0)89 5902 -502
Zoologisches Institut       FAX   :                -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
Luisenstr. 14
80333 Muenchen
Germany