Building T7 promoters onto primers

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Mon Sep 21 08:12:03 EST 1998


On Mon, 21 Sep 1998 12:51:34 +0100,
    Dr. Duncan Clark (duncan at nospam.demon.co.uk) wrote:
>I know I can just stick a T7 promoter sequence on the 5' end of one of
>my primers but is there any additional extra sequence after the promoter
>that would aid/increase the amount of RNA produced? Just wondering. I'll
>probably use an Ambion or Epicentre transcription kit.

I did this a few years ago  to produce RNAs corresponding to the
5' end of retroviral RNAs, and it works quite well.  My recollection
is that I used just a 17 bp T7 promoter, and ensured that the +1
residue was a "G".

There is a good discussion of the template requirements for T7 RNA
polymerase in a methods in enzymology chapter by Milligan and
Uhlenbeck, which I have listed below.

Milligan, J.F., and Unlenbeck, O.C. (1989) Synthesis of small RNAs
using T7 RNA polymerase. Methods in Enzymology 180:51-62

Ashok
-- 
Ashok Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
aiyar at ebv.oncology.wisc.edu



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