Annealing of two complementary circular SS DNAs

Ken Howe howe at DARWIN.UCSC.EDU
Fri Aug 16 10:21:27 EST 1996


Lloyd,
Two nonconcatenated (unlinked) circular ssDNAs may be partially
annealed, but you'll have a problem with supercoiling caused by the
helical nature of the duplex.  Although I've never worked with such
things and not knowing if they're even available, this next suggestion
may not be of any use: I suppose the addition a topoisomerase to your
annealed DNAs might allow for complete closed circular DNA helix to
form.

The obvious thing, assuming these aren't unusual derivatives of ssDNA,
is to anneal a primer to one of the two circles and extend/ligate to get
the second strand.

Just my thoughts...

On Fri, 16 Aug 1996, Lloyd Graham wrote:

> Date: Fri, 16 Aug 1996 02:01:29 GMT
> From: Lloyd Graham <graham at dbe.csiro.au>
> To: methods at net.bio.net
> Subject: Annealing of two complementary circular SS DNAs
>
> A procedure I am conducting will generate two closed circular
> single-stranded DNAs which are perfectly complementary to each other. I
> would like to anneal them together to generate a double-stranded DNA (a
> 3.6kb plasmid). Is it simply a matter of heating to 95'C and allowing them
> to cool slowly, or will their circular nature complicate things?
>
> Ultimately I just want to transform E.coli with the resulting plasmid.
> Perhaps it's not even necessary for me to do an annealing step - does
> anybody know if transforming with single-stranded plasmid DNA is
> effective?
>
> Many thanks,
>
> Lloyd Graham
> CSIRO Div. Biomol. Eng., Australia
>
>

                     _______________________________________

                                  Kenneth Howe
                     Center for the Molecular Biology of RNA
                             Department of MCD Biology
                             University of California
                              Santa Cruz, CA  95064
                     _______________________________________

                          e-mail: howe at darwin.ucsc.edu

               http://www-biology.ucsc.edu/people/areslab/ken.html




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