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In-vitro RNA transcripts using linearised transcripts, help.

Edwin ten Dam ebtd1 at cam.ac.uk
Sun Dec 10 11:16:35 EST 1995


In article <4aatb3$502 at dingo.cc.uq.oz.au>, forrest at biosci.uq.oz.au
(Alistair Forrest) wrote:

>G'day All,
>        I'm doing some In-vitro T7 transcripts. The uncut plasmid DNA
>works great but the linearised template is producing nothing.

Hmmm. Either your cutting buffer/enzyme/BSA? is contaminated with RNase
(try a fresh tube of each), You shouldn't add any RNase to the digest
(Don't worry about RNA from your DNA prep, just incubate longer with more
enzyme), or maybe you've got a restriction site close to the T7 promotor.
Try with another restriction enzyme. Phenol/Chloroform folowed bu EtOH ppt
should work, even on miniprep DNA. You could add a G50 spun column step,
but I find it's usually not needed. Good luck!

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Edwin ten Dam                                  ebtd1 at cam.ac.uk
Div. Virology, Univ. Cambridge      dam_e at rulgca.leidenuniv.nl
Tennis Court Road                       phone: +44 1223 336918
Cambridge CB2 1QP, UK                     fax: +44 1223 336926

"There are obviously many things which we do not understand, and
may never be able to." [Leela, Ship Operations AI, Marathon]



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