problem synthesizing full-length riboprobe

Stephan Spencer sspencer at rhino.bocklabs.wisc.edu
Tue Jan 25 12:42:25 EST 1994


I am doing an RNAse protection assay. The ifn-gamma riboprobe I obtained
most of the time is not full-length, which is important for this assay.
I am sure the template is clean and linearized completely as other templates
I used.  All others gave discrete full-length bands except this one.
So I have to gell-purify this probe but the yield is low and the procedure
is a pain. I would like to know if someone knows the conditions for the
in vitro transcription reaction which would appropropriate for this
template (ifn-gamma).  There are several things I could do but it costs
money and takes time. Could someone suggest which one, if any, would be
appropriate? The ideas I have are PCR clone another region of this gene,
or change the concentration of the nucleotides in the reaction (37.5 
micromolar UTP, 0.5 millimolar ATP, GTP, CTP, and some P-32 labelled UTP).

Please respond either to the group or to the following email address:
       ma at biochem.wisc.edu

Thank you,
Yan Ma

--- Posted by Stephan Spencer.  Please do not reply to my email address.
    Reply to ma at biochem.wisc.edu.




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