in vitro transcription problem
pnorton at lac.jci.tju.edu
Fri Sep 29 09:32:11 EST 1995
In article <442g3r$ca8 at news.acns.nwu.edu>, z-scott at nwu.edu (Rizaldy P
> During extraction (alkaline or thermal lysis preps), plasmids
> denature. Some of these molecules however do not fall
> back perfectly as double helices; the two strands of these
> molecules may fall out of register and persist simply as
> concatenated single strands. These concantenates would be
> resistant to restriction endonucleases and could be missed
> out on a minigel check since they do not stain with ethidium
> bromide as well as paired helices. Nevertheless, these single
> stranded molecules remain as templates for RNA polymerases
> and would provide transcripts past the supposed nicking sites
> of the restriction enzymes that were used. There are two
Interesting thought, but it has been found that the promoter region
must be double stranded, at least for T7 RNA polymerase. The template can
be single-stranded. Thus, denatured SS DNA would not be expected to give
rise to any transcripts. I don't know about T3 or SP6 polymerases, but I
suspect that they would have similar requirements.
Ref: Milligan et al, NAR 15:8783 (1987).
For the record, I believe the most likely explanation for the original
poster's problem was already offered: use of an enzyme that leaves a 3'
overhang for linearization can result in hairpins.
Pamela A. Norton, Ph.D. Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107 p_norton at lac.jci.tju.edu
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