in vitro transcription problem

Pamela Norton 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
Scott) wrote:

> 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. 

            Pam Norton

-- 
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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