How long in vitro transcripts?

Heinz J. Schaefers schaefers at sun.XuchcX.edu
Wed Mar 18 16:22:35 EST 1998



brett wrote:

> >Hello netters,
> >Does anyone have experience in producing in vitro transcripts 4-5kb
> >long? We are considering this for a cDNA almost 5000 bases long, with
> >rather long 5' and 3' untranslated regions (the coding region is "only"
> >3000 bases). We need to transcribe the whole length in order to look for
> >the accessible sites for antisense oligos. I would greatly appreciate
> >any suggestions or comments!
> >Marek Treiman
> This should be no problem. RNA virologists have been transcribing full
> length infectious RNA up to 13kb for some time now.  My only advice is
> to not buy a kit - especially one guaranteeing high yields (ie MegaScript).
> Their idea of RNase-free means that the RNase is included at no extra charge.
> Plus you will have an easier time if you do have to troubleshoot if
> you make everything yourself. Happy transcribing.
> Brett Lindenbach

Hi,
i use the promega kit "riboprobe system sp6 / t7".
no problem at all, i did transcripts up to 4.5 kb length.
as recommended in the promega manual for longer transcripts, i did the reaction
at 30 degrees centigrade instead of 37 degrees.

Heinz
(no affiliation with promega!)
--
Heinz J. Schaefers, Ph.D.
University of Connecticut Health Center, Dept. of Physiology, Farmington, CT
schaefers at sun.XuchcX.edu (antispam: remove both X)





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