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...
> Hello
>
>  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?
>
>  Arox

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:

(dT)20.V.N

where V = either A, C or G, and N = any base.

You can order it from your favourite oligo provider (I use MWG).

Jose




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