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In-vitro RNA transcripts using linearised transcripts, help.

Steve Hartson shartson at BMB-FS1.BIOCHEM.OKSTATE.EDU
Wed Dec 13 09:23:14 EST 1995


Alistair,

you wrote:

        "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

                                'luck,

                                        Steve

///////////////////////////////////////////////////////////////////////

Steve Hartson
Dept. Biochem. and Mol. Biol.
246 NRC
Okla. St. U.
Stillwater, OK  74074  USA
shartson at bmb-fs1.biochem.okstate.edu





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