T7 transcription of oligos
pd1 at mole.bio.cam.ac.uk
Mon Jun 17 09:49:11 EST 1996
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
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.
Division of Virology
Department of Pathology
University of Cambridge
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