How long in vitro transcripts?
Heinz J. Schaefers
schaefers at sun.XuchcX.edu
Wed Mar 18 16:22:35 EST 1998
> >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
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.
(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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