I'm doing some In-vitro T7 transcripts. The uncut plasmid DNA
works great but the linearised template is producing nothing.
I originally just tried phenol chloroforming the digest once fully cut and
then ethanol precipitating using sodium acetate. This gave me nothing so I
guessed I'd either got and RNAse in during the digest and clean up or that
there was too much salt being carried over to the end reaction. The
restriction digest was in high salt buffer. To try to overcome this I set
up a small reaction digest and then tried taking a small aliquot of the
digest and in-vitro transcribing that after dilution in the correct
buffer. I also tried ammonium acetate precipitation of another aliquot of
the sample (after phenol chloroforming) but neither of these approaches
gave me in-vitro transcripts for the cut samples.
Does anyone have any ideas of how to get around this. All the
transcription reactions are set up in depc treated water and RNAsin is
included in the reaction.
Thanks for any suggestions,
Alistair Forrest (Phd student)