T7 PCR primers to synthesise RNA standards

Glenn Knight glenn.knight at lahey.hitchcock.org
Thu Dec 18 19:01:37 EST 1997


liz wrote in message <34996102.4FB4 at mx3.usuhs.mil>...
>Hi! I'm looking for advice concerning the construction of RNA standards for
>quantitative RT-PCR.


I've been using the PCR method to incorporate T7 sequences into
transcription templates for several years with much greater and more
consistent success than with linearized plasmids. It is a quick way to
generate sufficient template DNA within a couple of hours. The PCR product
can be used directly in the transcription reaction without purification.
Also, the T7 promoter sequences in some of the plasmid vectors are not very
efficient so be sure to use a good promoter sequence in the primer. There
are a few references on the effects of sequence variation in the promoter
and the efficiency of transcription that I can dig out, if you're
interested.
Glenn





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