Detection of ss DNA intermediate

Richard Koepsel rrk1+ at pitt.edu
Tue Jul 22 08:26:51 EST 1997


Stephen Billington wrote:
> 
> Hi all
> 
> I am currently attempting to show that a plasmid replicates by rolling
> circle replication by detecting single stranded intermediate. This is
> usually done by Southern blot without denaturing the DNA prior to
> transfer. However, everytime I try to do this I get double stranded DNA
> transferring to the blot. I know this because my control which is just a
> pUC based plasmid lights up in the Southern. Does anyone have any
> experience with this sort of thing and can perhaps help me? This is
> particularly frustrating as it seems to be a standard procedure and I
> have a manuscript waiting for this one experiment.
> 
> sjb

Stephen,
Years ago we used a number of techniques to show single stranded plasmid
replication intermediates (look at papers from Saleem A. Khan's group
from the mid to late 80's).  I would suggest that you probe your
southerns with strand specific probes.  We used to end label fragments
strand seperate on denaturing gels and cut out each strand seperately. 
There are currently better techniques such as in vitro transcription
(using something like the pGEM plasmids from Promega) or perhaps
asymetric PCR to generate strand specific probes.  Probing with one
strand then stripping the blot and probing with the other should provide
a nice internal control.
Good luck,
Rick Koepsel



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