Clore Laboratory CLORELAB at clorelab.demon.co.uk
Fri Dec 6 08:51:31 EST 1996

Dear netter
        I have had an odd experience with my riboprobe synthesis. I hope
someone from the scientific community will comment on this. I have been
preparing riboprobes from at least 6 different sequences (cDNAs), by
using the following procedure; I either obtain dscDNA (400-600 bp) by
PCR amplification of vector inserts or from cDNA libraries. The PCR
fragments are then ligated into a pCR-TRAP system (blunt-end ligation).
Here, I expect that the orientation of the ligated inserts is random (is
this correct). Then I reamplify the fragments, but now using primers
that contain either the T7 core element for the T7 pol. or the SP6 core
element for the SP6 pol.. The resulting templates are then used to
synthesize either the T7 cRNA strand or the SP6 strand. This is a
conventional procedure and the whole method works perfectly well,
leading to high yield of cRNA. Naturally, you would expect the antisense
to be 50% T7 and 50% SP6 derived, however 6 different cRNAs (6 T7 cRNA
and 6 SP6 cRNA) have been produced so far and only the SP6 cRNAs are
antisense to their cognate mRNA. Is this a coincidence or is there some
mechanistic explanation for this.  Can someone comment.

Can anyone comment on this
V. Emilsson
Clore Laboratory

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