Building T7 promoters onto primers

ELADER at AMBION.COM ELADER at AMBION.COM
Wed Sep 23 10:41:25 EST 1998


In article <LT7aVCAG3jB2Ew52 at genesys.demon.co.uk>,
  "Dr. Duncan Clark" <duncan at genesys.demon.co.uk> wrote:
> Hi Folks,
>
> I need to run a transcript off a 1kb PCR product purely as a means to
> generate RNA as a test system for an RT reaction.
>
> 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.
>

5'-TAATACGACTCACTATAGGG(AGGAGG)  Add this to the 5' end of your 5' primer to
make a template for sense strand RNA. You can use the PCR product in the
transcription reaction without further purification.The first transcribed
base is the middle G in the triplet before the parenthesis. The bases in the
parenthesis are optional . We add them so there is no C or U transcribed in
the first couple of bases. When making a high specific activity C or U
labeled probe, this helps avoid premature termination (no Clinton jokes
please).

Eric

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