"I'm doing some In-vitro T7 transcripts. The uncut plasmid DNA
works great but the linearised template is producing nothing. (deleted)"
My thoughts: I have read in more than one place (but I can't remember
where) that T7 is very inefficient on linear templates. Thus, I propose
that you have an inefficient polymerase whose yield is below limits of
precipitation and/or detection. This may be compounded by low template
concentration, since "cut" template is typically phenol extracted and
recovery after phenol extraction/EtOH ppn is only 50% (in my hands).
My suggestiosn: 1) confirm DNA concentration in IVT rxn
2) after IVT, run aliquot of rxn on AGE *without* cleanup
do +/- polymerase controls to ID RNA product
3) get fresh polymerase and use 2-3X "normal" volumes
Dept. Biochem. and Mol. Biol.
Okla. St. U.
Stillwater, OK 74074 USA
shartson at bmb-fs1.biochem.okstate.edu