Primer EXTension

Antonin Tutter atutter at jeeves.ucsd.edu
Mon Jan 13 21:52:38 EST 1997


darren cikaluk wrote:
> 
> I am looking for a primer extension protocol that someone has had great
> success with.
> I know of manniatus and the red books' protocol but If any one else ahs
> any tips on this procedure that would be great.


Darren,

our lab uses primer extension (PE) routinely.  The success depends
largely on what sort of template you are using (we use in vitro
transcribed RNA) and the design of the primers (we use a 25-mer,
anti-sense, 5' labeled), so it would be helpful to know exactly what you
would like to assay for using PE before making suggestions.  PE is a
great way to quantitatively assay transcription initiation rates from a
promoter, and that is what we use it for.

One important thing to keep in mind is that the primer extension product
that you design a primer for should not be more than ~150 bp, because
reverse transcriptase has poor processivity after the first 100 - 150 bp
or so.

Let me know how you would like to use it and I can either post to the
newsgroup or e-mail you our protocol.


Anton Tutter
atutter at jeeves.ucsd.edu



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