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In-vitro RNA transcripts using linearised transcripts, help.

pgegen at rnaworld.bio.ukans.edu pgegen at rnaworld.bio.ukans.edu
Mon Dec 11 20:10:54 EST 1995


In <ebtd1-1012951616350001 at mac26.vrl.path.cam.ac.uk>, ebtd1 at cam.ac.uk (Edwin ten Dam) writes:
>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!

We have found that poor transcription yields can usually be improved either by extraction twice with phenol:chloroform:isoamyl alcohol (25:24:1) and twice with chloroform:isoamyl (24:1), followed by EtOH pptn, OR by simply reprecipitating the DNA with 2.5 vol ammonium acetate followed by 2 to 2.5 vol EtOH. Both of these treatments assume that the DNA is free of nucleases. (Crystalline BSA, sometimes even acetylated BSA, is usually heavily contaminated with RNases.) If NOT, then the proteinase K digestion i
s a must!

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