T7 transcription of oligos

Paul Digard pd1 at mole.bio.cam.ac.uk
Mon Jun 17 09:49:11 EST 1996


Hi All

We've been trying to synthesise short (about 12 - 30 base) RNAs
by in vitro transcription of annealed oligos, one containing a 
minimal T7 promoter, the other containing the complement to the 
promoter, and a single strand extension specifying the transcript 
sequence.  

This technique works for others, but we've been getting very poor 
results; basically a one base ladder up to the predicted length, but 
also beyond. Today's gel had a ladder up to 30 (the expected size, 
but barely more intense than any other product), but then up to more 
than 70 nucleotides.

We've gel purified the oligos.  The transcripts are meant to be 
devoid of secondary structure (very U rich in fact), and are heated 
to 90 C before being electrophoresed on pre-run polyacryl/urea gels, 
so we're completely mystified as to what the longer species are.  
We've tried a few different buffer compositions, but haven't yet 
systematically altered pH, MgCl2 or NTP concentrations.

So, does anybody have any suggestions?

Thanks in advance.


Paul Digard

Division of Virology
Department of Pathology
University of Cambridge



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