Reverse Transcriptase Assay
Jose de las Heras
josenet at tiscali.co.uk
Thu May 4 14:16:52 EST 2006
"Kamng'ona A W" <awkamngona at yahoo.co.uk> wrote in message
news:mailman.822.1146749264.16885.methods at net.bio.net...
> I am carrying out a simple reverse transcriptase assay using Poly U
> template (1000bp) and Poly A primer (20 mer).
> This primer would be expected to bind non-specifically to the template. I
> would however like to have the primer bind specifiacally to one end of the
> template so tha I achieve a full length cDNA synthesis.
> What should I do?
I'm not sure I understood correctly, but if you're talking about amplifying
your cDNA from the 5' end where the polyA sits, use "anchored" oligos.
Typically, to amplify your polyA-containing mRNA, you'd use a mixture of
oligos, say about 20"T" plus another two, of random sequence, to anchor it
to the end of the mRNA. Something like:
where V = either A, C or G, and N = any base.
You can order it from your favourite oligo provider (I use MWG).
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